Method to use viral and host methylation markers for cervical cancer screening and triage in liquid prep, serum/plasma, and urine: PCR and sequencing based process methods

ABSTRACT

Methods and kits for triaging a human papillomavirus (HPV)-positive woman into colposcopy are disclosed. The methods comprise determining the promoter methylation level of the promoter regions of a group of genes (e.g., viral and host genes) that exhibit increased promoter methylation in women having CIN2+ lesions as compared to women having no intraepithelial lesions or malignancy (NILM).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. § 371 U.S. national entry ofInternational Application PCT/US2016/044225, having an internationalfiling date of Jul. 27, 2016, which claims the benefit of U.S.Provisional Application No. 62/197,306, filed Jul. 27, 2015, the contentof each of the aforementioned applications is herein incorporated byreference in their entirety.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant numbersCA084986, and CA164092, awarded by the National Institutes of Health.The government has certain rights in the invention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

This application contains a sequence listing. It has been submittedelectronically via EFS-Web as an ASCII text file entitled“P13649-03_ST25”. The sequence listing is 2,335 bytes in size, and wascreated on Aug. 2, 2018. It is hereby incorporated by reference in itsentirety.

BACKGROUND

Cervical cancer screening is undergoing a major transformation with theadoption of testing for the presence of oncogenic human papilloma virus(HPV) types (1, 2). Persistent HPV infection of the cervical epitheliumis a rare event because most infections are usually cleared withouttreatment (3). Yet a small percentage of HPV infections are associatedto progression from low-grade squamous intraepithelial lesions (LSIL) tocervical intraepithelial neoplasia grade 3 (CIN3) lesions (4). In fact,persistent infection with at least one of the 13 carcinogenic types fromthe alphapapillomavirus genus (HPV16, 18, 31, 33, 35, 39, 45, 51, 52,56, 58, 59, 68) has been linked to cervical cancer (5). Close to 30% ofCIN3 lesions progress to cervical cancer, but there are no agreed uponclinical tests of progression to cervical cancer (6, 7).

The new cervical cancer screening guidelines in the United Statesrecommend HPV co-testing with Pap among women 30 years and older (8).However, clinical management for HPV-positive/Pap-negative women is notfirmly established (9, 10). HPV has recently been shown to be a betterindicator of cervical cancer risk than the Pap test (11). But even whenco-testing with Pap and HPV has higher sensitivity and specificity thaneach separate test, together they cannot predict who will progress tocervical carcinoma (12, 13).

This led to the search of molecular markers of risk to progression tocervical cancer. HPV DNA methylation, like other DNA viruses, iscorrelated to progression to cancer (14). This seminal finding led tosubsequent studies suggesting that detection of methylated HPV DNA maydistinguish women with cervical intraepithelial neoplasia grade 2-3(CIN2+) from women with an oncogenic HPV type infection who show noevidence of CIN2+ (15-19). In parallel, other groups examined theassociation between host DNA methylation and cervical cancer (20-23).Recent studies, have reported a positive association between CIN2+ andmethylation of CpG sites in host and viral DNA isolated from liquidcytology samples (19, 20, 24).

Recently published reports have also explored the use of urine-basedhigh risk HPV testing, as an alternative approach to liquid cytology forcervical cancer screening, in an attempt to identify less invasivecervical cancer screening technologies (25-27). Most of the studies havefailed to attain clinical usefulness as they are limited by poorsensitivity, inappropriate protocols for DNA extraction from circulatingDNA in urine, small sample size, and the limit of detection of the HPVassays utilized (28-30). Most of the studies also fail to recognize theimportance of using DNA isolation methods optimized to enrich forfragmented circulating cell-free DNA (ccfDNA) that crosses the kidneysand can be obtained in urine (31).

There is growing evidence that circulating short human, viral, andbacterial DNA fragments from dying cells throughout the body,approximately 150-250 bases long on average, pass through the renalbarrier and appear in urine as Transrenal DNA (TrDNA)(32) or ccfDNA.ccfDNA has been proposed as a tool for non-invasive prenatal monitoring,infectious disease monitoring, and tumor response monitoring (33-35).Recently, a capillary electrophoresis ccfDNA test that targets the E1region of the HPV genome for the detection of high risk HPV demonstratedhigh sensitivity and modest specificity for urine-based detection ofcervical pre-cancerous lesions (36).

Urine samples tested by the ccfDNA HPV capillary electrophoresis testhad high concordance with corresponding cervical and urine samplestested by the widely used Linear Array HPV Genotyping Test (LA-HPV)(37).However, the ccfDNA capillary electrophoresis HPV test is notquantitative, can only detect the presence or absence of high risk HPVand, similar to previously published urine based HPV tests, has limitedspecificity. None of the high-risk HPV urine-based reports usesequencing based approaches to quantify multiple HPV types, nor includemethylated markers in their workflow to improve the sensitivity andspecificity of the ccfDNA test.

SUMMARY

The practice of the present invention will typically employ, unlessotherwise indicated, conventional techniques of cell biology, cellculture, molecular biology, transgenic biology, microbiology,recombinant nucleic acid (e.g., DNA) technology, immunology, and RNAinterference (RNAi) which are within the skill of the art. Non-limitingdescriptions of certain of these techniques are found in the followingpublications: Ausubel, F., et al., (eds.), Current Protocols inMolecular Biology, Current Protocols in Immunology, Current Protocols inProtein Science, and Current Protocols in Cell Biology, all John Wiley &Sons, N.Y., edition as of December 2008; Sambrook, Russell, andSambrook, Molecular Cloning. A Laboratory Manual, 3^(rd) ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, 2001; Harlow, E. andLane, D., Antibodies—A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, 1988; Freshney, R. I., “Culture of AnimalCells, A Manual of Basic Technique”, 5th ed., John Wiley & Sons,Hoboken, N.J., 2005. Non-limiting information regarding therapeuticagents and human diseases is found in Goodman and Gilman's ThePharmacological Basis of Therapeutics, 11th Ed., McGraw Hill, 2005,Katzung, B. (ed.) Basic and Clinical Pharmacology, McGraw-Hill/Appleton& Lange 10^(th) ed. (2006) or 11th edition (July 2009). Non-limitinginformation regarding genes and genetic disorders is found in McKusick,V. A.: Mendelian Inheritance in Man. A Catalog of Human Genes andGenetic Disorders. Baltimore: Johns Hopkins University Press, 1998 (12thedition) or the more recent online database: Online MendelianInheritance in Man, OMIM™. McKusick-Nathans Institute of GeneticMedicine, Johns Hopkins University (Baltimore, Md.) and National Centerfor Biotechnology Information, National Library of Medicine (Bethesda,Md.), as of May 1, 2010, World Wide Web URL:http://www.ncbi.nlm.nih.gov/omim/ and in Online Mendelian Inheritance inAnimals (OMIA), a database of genes, inherited disorders and traits inanimal species (other than human and mouse), athttp://omia.angis.org.au/contact.shtml.

In an aspect, the presently disclosed subject matter provides a methodfor triaging a human papillomavirus (HPV)-positive woman intocolposcopy, the method comprising: (a) selecting a HPV-positive womantesting positive for one or more high risk types of HPV; (b) obtainingnucleic acid from a test sample from the selected HPV-positive woman;(c) determining in the nucleic acid from the test sample of the selectedHPV-positive woman a promoter methylation level of the promoter regionsof a group of genes that exhibit increased promoter methylation in womenhaving CIN2+ lesions as compared to women having no intraepitheliallesions or malignancy (NILM); and (d) triaging the HPV-positive womaninto colposcopy when the level of promoter methylation of the group ofgenes is increased relative to the level of promoter methylation of thegroup of genes in a reference sample obtained from women having NILM.

In an aspect, the presently disclosed subject matter provides a methodfor triaging a human papillomavirus (HPV)-positive woman intocolposcopy, the method comprising: (a) selecting a HPV-positive womantesting positive for one or more high risk types of HPV; (b) providing akit that comprises the probes and/or primers needed for determining inthe nucleic acid from a test sample of the selected HPV-positive woman apromoter methylation level of the promoter regions of a group of genesthat exhibit increased promoter methylation in women having CIN2+lesions as compared to women having no intraepithelial lesions ormalignancy (NILM); (c) obtaining nucleic acid from the test sample fromthe selected HPV-positive woman; (d) determining the promotermethylation level in the nucleic acid from the test sample of theselected HPV-positive woman using the provided kit; and (e) triaging theHPV-positive woman into colposcopy when the level of promotermethylation of the group of genes is increased relative to the level ofpromoter methylation of the group of genes in a reference sampleobtained from women having NILM.

In an aspect, the presently disclosed subject matter provides a kit fortriaging a human papillomavirus (HPV)-positive woman into colposcopy,the kit comprising: (a) a container containing primers and/or probesspecific for a group of genes that exhibit increased promotermethylation in women having CIN2+ lesions as compared to women having nointraepithelial lesions or malignancy (NILM); and (b) instructions foruse of the primers and/or probes in triaging a human papillomavirus(HPV)-positive woman into colposcopy.

Certain aspects of the presently disclosed subject matter having beenstated hereinabove, which are addressed in whole or in part by thepresently disclosed subject matter, other aspects will become evident asthe description proceeds when taken in connection with the accompanyingExamples and Figures as best described herein below.

BRIEF DESCRIPTION OF THE FIGURES

Having thus described the presently disclosed subject matter in generalterms, reference will now be made to the accompanying Figures, which arenot necessarily drawn to scale, and wherein:

FIG. 1A shows a description of the method used. Exemplary steps 1-9 asshown include liquid prep and or urine sample is collected and sent tothe lab, DNA isolation, human and HPV DNA sequence capture, bisulfitetreatment of human and HPV DNA (or enzymatic restriction with CGsensitive enzymes), sequencing, HPV genotyping to identify high riskstrains, human and HPV DNA methylation analyses to identifydifferentially methylated regions (DMRs), identify premalignantlesions/assess cervical cancer risk/assess risk of progression to CIN2+,and deliverables: cervical cancer screening and/or triage results. FIG.1B shows the project workflow, for example, discovery was performed incervical cancer biopsy samples and cervical brush epithelium from normalcontrols. Nimblegen 385K CpG plus Promoter oligonucleotide arrays wereused to hybridize samples enriched with Methylated DNAImmunoprecipitation (MeDIP). Promoter methylation of ZNF516, INTS1 andFKBP6 discriminated between normal and cancer samples. The panel wasValidated on cervical brush samples from patents with CervicalIntraepithelial Neoplasia 2-3 (CIN2+) compared to patents with NoIntraepithelial Lesions or Malignancy (NILM) (n=211). Promotermethylation of ZNF516, INTS1 and FKBP6, the host gene panel, togetherwith HPV16-L1 DNA methylation discriminated between CIN2+ and NILM onliquid prep samples (n=67). Custom sequence capture probes were designedto capture the high-risk HPV genome and methylome, before performinglibrary prep for massively parallel Next-Generation Sequencing. TheccfDNA HPV16-L1 methylation is tested in urine, and plasma samples;

FIG. 2A shows ROC curves of FKBP6 INTS1 ZNF516 promoter methylation,comparing CIN2-3 vs NILM in cervical brush samples from Chile (n=126).FIG. 2B shows ROC curve of a combined molecular panel integrated byFKBP6 INTS1 ZNF516 promoter methylation, comparing CIN2-3 vs NILM incervical brush samples from Chile (n=126);

FIG. 3A shows ROC curves of FKBP6, INTS1, ZNF516 promoter methylationand HPV16-L1 methylation, comparing CIN2-3 vs NILM in liquid-basedcytology samples (n=67). FIG. 3B shows ROC curve of a combined molecularpanel integrated by FKBP6, INTS1, ZNF516 promoter methylation andHPV16-L1 methylation, comparing CIN2-3 vs NILM in liquid-based cytologysamples. (n=67);

FIG. 4 shows amplification curves for the HPV ccfDNA-qPCR assay onccfDNA from women with Cervical Intraepithelial Neoplasia 1 (CIN1),Cervical Intraepithelial Neoplasia 2-3 (CIN2-3) and women with noIntraepithelial Lesions or Neoplasia (NILM). ccfDNA samples wereanalyzed for high risk HPV DNA using our HPV ccfDNA SYBR green qPCRassay. HeLa genomic DNA was included as a positive control. Ct rangesare variable, but are in a similar range as the genomic control. NILMsamples did not amplify;

FIG. 5 shows sequence capture of methylated high risk HPV.

FIG. 6A shows ROC curves for HPV16-L1 methylation and ZNF516, FKBP6 andINTS1 promoter methylation in plasma ccfDNA samples from patientsdiagnosed with CIN2+ and NILM, with area under ROC curve=0.8075. FIG. 6Bshows ROC curves for HPV16-L1 methylation and ZNF516, FKBP6 and INTS1promoter methylation in urine ccfDNA samples from patients diagnosedwith CIN2+ and NILM, with area under ROC curve=0.8611;

FIG. 7 shows a custom-designed pool of HPV-specific dual sequencecapture probes was used for library amplification and target selectionin ccfDNAs (Roche/NimbleGen SeqCap EZ Choice Library). Using a SYBRgreen qPCR assay, an HPV E1 region common to thirteen high-risk HPVtypes was amplified;

FIG. 8 shows an amplification plot demonstrating the successful HPVamplification and enrichment obtained with dual sequence capture. DeltaCt values for HeLa (pink/red) and CSCC7 (purples) were 14.88 and 12.66respectively. Based on an estimated efficiency for the assay, theapproximate fold enrichment was greater than 1700;

FIG. 9 shows Pre- and Post-Capture HPV qPCR results for five ccfDNAsamples together with HeLa genomic control (dark blue) and no DNAcontrols (purple);

FIG. 10 shows a profiling of sample TrDNA-456 by tiered read mapping tovarious databases, the graph indicates the percentage of reads that mapto thirteen (13) HPV types, human, bacteria and unknown genomes afterprofiling reads of the clinical sample TrDNA-456 by tiered read mapping;

FIG. 11 shows box plots showing the distribution of CpG methylationlevels per sample after aligning to HPV16 (left) and HPV16-L1 region(right). The CpG methylation median in the clinical samples issignificantly higher than in the cell lines and higher in urine ccfDNAfrom the CIN3 than from the CIN1 clinical sample (p<0.05), as expected;

FIG. 12 shows Scatter plots of HPV16-L1 methylation in urine ccfDNA.HPV16-L1 qMSP methylation can discriminate bisulfate treated urineccfDNA from patients with normal cytology (n=10) from women withdysplastic cytology and premalignant cervical lesions (ASCUS n=8; CIN1n=3; CIN2+ n=3) with 100% Sensitivity and Specificity;

FIG. 13 shows circulating cell-free Renal DNA (ccfDNA) isolation resultsin the table. The table shows the comparison between DNA concentrationobtained by isolating DNA from urine from the same participantsutilizing two different extraction methods: Phenol chloroform (PC) andccfDNA isolation methods; the illustrations shows a schematic of oneconcept of how ccfDNA enters the blood and urine of the subject;

FIG. 14 shows a table comprising dual sequence capture of high risk HPVin ccfDNA aligned to all high risk HPV types and low risk HPV types fromPaVE database (top) and box plots of dual sequence capture of high riskHPV in ccfDNA aligned to all high risk HPV types and low risk HPV types(bottom); and

FIG. 15 shows pairwise alignment of the HPV16 genome with reads obtainedfrom urine ccfDNA from seven clinical samples: TrDNA-445, TrDNA-455,TrDNA-456, TrDNA-481, TrDNA-504, TrDNA-513, and TrDNA-571. The“close-up” HPV DNA computes and displays nucleotide-level (close-up′)multiple alignments of sequences in a 1 Kb region starting at auser-specified address or gene in the reference genome.

DETAILED DESCRIPTION

The presently disclosed subject matter now will be described more fullyhereinafter with reference to the accompanying Figures, in which some,but not all embodiments of the inventions are shown. Like numbers referto like elements throughout. The presently disclosed subject matter maybe embodied in many different forms and should not be construed aslimited to the embodiments set forth herein; rather, these embodimentsare provided so that this disclosure will satisfy applicable legalrequirements. Indeed, many modifications and other embodiments of thepresently disclosed subject matter set forth herein will come to mind toone skilled in the art to which the presently disclosed subject matterpertains having the benefit of the teachings presented in the foregoingdescriptions and the associated Figures. Therefore, it is to beunderstood that the presently disclosed subject matter is not to belimited to the specific embodiments disclosed and that modifications andother embodiments are intended to be included within the scope of theappended claims.

The presently disclosed subject matter provides methods and kits fortriaging a human papillomavirus (HPV)-positive woman into colposcopy. Ithas been found that a group of genes exhibit increased promotermethylation in women having CIN2+ lesions as compared to women having nointraepithelial lesions or malignancy (NILM). The presently disclosedmethods provide a triage test that can discriminate between those womenwith no intraepithelial lesions or malignancy (NILM) and women withabnormal cervical biopsies (CIN1, CIN2, CIN3), carcinoma in-situ, andcervical cancer.

Rapid advances in genomic technologies and TCGA have revealed extensive,previously unsuspected complexity in human cancers. This complexity hasled to calls for “precision medicine” to address the underlying complexgenetic and epigenetic processes that underlie somatic cell evolution tomalignancy. The present inventive methods take a significant stepforward to improve precision medicine based on DNA sequence changes inmethylation and mutation of the somatic genome of the patient and HPV.The inventive methods are anchored in the phases of biomarkerdevelopment as published by NCI EDRN several years ago. They alsoinclude cloud based approaches for generalized testing andimplementation of early detection strategies. Specific strategiesemployed include DNA sequence based assays such as quantitativeMethylation Specific PCR, bisulfate genomic sequencing, and NextGeneration Sequencing, all of which generate complex datasets reflectingthe complexity of neoplastic evolution in human tissues. The inventivemethods also propose testing urine samples using methods that optimizeDNA isolation to enrich for cell free fragmented DNA (cffDNA) that isexcreted in the urine, which opens a novel approach to early detection.The cloud based tools proposed in the methods could eventually be usedby patients, providers, researchers and even insurance personnel. Thistype of “precision medicine” approach based on the actual DNAmethylation and DNA nucleotide changes in the evolving virus andneoplasm of necessity vastly increase the complexity of the data.

I. Methods for Triaging a Human Papillomavirus (HPV)-Positive Woman intoColposcopy

In some embodiments, the presently disclosed subject matter provides amethod for triaging a human papillomavirus (HPV)-positive woman intocolposcopy, the method comprising: (a) selecting a HPV-positive womantesting positive for one or more high risk types of HPV; (b) obtainingnucleic acid from a test sample from the selected HPV-positive woman;(c) determining in the nucleic acid from the test sample of the selectedHPV-positive woman a promoter methylation level of the promoter regionsof a group of genes that exhibit increased promoter methylation in womenhaving CIN2+ lesions as compared to women having no intraepitheliallesions or malignancy (NILM); and (d) triaging the HPV-positive womaninto colposcopy when the level of promoter methylation of the group ofgenes is increased relative to the level of promoter methylation of thegroup of genes in a reference sample obtained from women having NILM.

As used herein, the term “colposcopy” refers to a medical procedure thatuses a device, such as a magnifying device, to view the cervix of awoman. If a problem is seen during a colposcopy, a small tissue sampleor biopsy can be taken from the cervix or from inside of the opening ofthe cervix. A goal of a colposcopy is to detect cervical dysplasia, suchas intraepithelial lesions, premalignant cervical neoplasia lesionsand/or cervical cancer lesions. As used herein, the term “triaging”refers to the sorting of and/or allocation of treatment to patients,particularly those women at risk for cervical cancer.

As used herein, a human papillomavirus (HPV)-positive woman or a womanwho tests positive for HPV is a woman who has been infected with HPV,particularly a high risk type of HPV. The term “high-risk HPV” as usedherein refers to those HPV types or strains that may progress toprecancerous lesions and invasive cancer, such as cervical cancer.Non-limiting examples of high-risk HPV strains that are known or thoughtto cause cervical cancer include HPV 16, 18, 26, 31, 33, 35, 39, 45, 51,52, 53, 56, 58, 59, 66, 68, 73, and 82. In some embodiments, one or morehigh risk types of HPV are selected from the group consisting of HPV16,HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58,HPV59, and HPV68. In some embodiments, a woman who has been infectedwith a high risk type of HPV is selected for triaging using thepresently disclosed methods. In some embodiments, the HPV-positive womanis further selected on the basis of abnormal cytology, such as a womanwho has cervical cells that do not appear normal in shape, size, and/orcomposition. In some embodiments, the abnormal cytology comprises lowsquamous intraepithelial lesions (LSIL). In some embodiments, theabnormal cytology comprises high squamous intraepithelial lesions(HSIL). In some embodiments, the HPV-positive woman does not haveabnormal cytology of cervical cells. In some embodiments, abnormalcytology is tested for by using a Papanicolaou test (Pap-test). In someembodiments, the HPV-positive woman has had a negative, positive, orinconclusive Pap smear.

In some embodiments, selecting the HPV-positive woman testing positivefor one or more high risk types of HPV comprises determining whether thenucleic acid is homologous to one or more high risk types of HPV. Insome embodiments, determining whether the nucleic acid is homologous toone or more high risk types of HPV comprises performing at least one HPVdetection assay selected from the group consisting of nucleic acidsequencing (e.g., Sanger sequencing, third generation sequencing, andthe like), PCR, a HPV genotyping assay, a microarray assay, and a mRNAbased assay. An example of a HPV detection assay is disclosed in PCTPatent Application No. PCT/US2014/019934, which is hereby incorporatedby reference in its entirety. Methods to determine homology between twonucleic acid sequences are well known in the art and any method that canbe used to determine homology can be used for the presently disclosedmethods.

In some embodiments, determining whether the nucleic acid is homologousto one or more high risk types of HPV comprises: (a) sequencing thenucleic acid to produce a nucleotide sequence; (b) performing a sequencealignment between the nucleotide sequence and the nucleotide sequence ofthe one or more high risk types of HPV; and (c) determining thepercentage sequence identity between the nucleotide sequence and thenucleotide sequence of the one or more high risk types of HPV.

“Sequence identity” or “identity” in the context of two nucleic acid orpolypeptide sequences includes reference to the residues in the twosequences which are the same when aligned for maximum correspondenceover a specified comparison window, and can take into considerationadditions, deletions and substitutions. When percentage of sequenceidentity is used in reference to proteins it is recognized that residuepositions which are not identical often differ by conservative aminoacid substitutions, where amino acid residues are substituted for otheramino acid residues with similar chemical properties (for example,charge or hydrophobicity) and therefore do not deleteriously change thefunctional properties of the molecule. Where sequences differ inconservative substitutions, the percent sequence identity may beadjusted upwards to correct for the conservative nature of thesubstitution. Sequences that differ by such conservative substitutionsare said to have sequence similarity. Approaches for making thisadjustment are well-known to those of skill in the art.

“Percentage of sequence identity” means the value determined bycomparing two optimally aligned sequences over a comparison window,wherein the portion of the polynucleotide sequence in the comparisonwindow may comprise additions, substitutions, or deletions (i.e., gaps)as compared to the reference sequence (which does not compriseadditions, substitutions, or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid base or amino acid residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the window of comparison and multiplying the result by 100to yield the percentage of sequence identity. Algorithmic programs forsequence comparisons and/or searching sequences against databases arewell known in the art, such as FASTA, BLAST, SAHA, MUMmer, AVID, CHAOS,QUASAR, and the like.

The terms “substantial identity” or “homologous” in their variousgrammatical forms in the context of polynucleotides means that apolynucleotide comprises a sequence that has a desired identity, forexample, at least about 60% or more identity, such as 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity compared toa reference sequence. Alignment programs using standard parameters canbe used to determine percent identity. In some embodiments, at least a60% sequence identity between the nucleotide sequence from the testsample and the nucleotide sequence of the one or more high risk types ofHPV means that at least one high risk HPV strain has been detected inthe woman.

In some embodiments, nucleic acid is obtained from a test sample fromthe selected HPV-positive woman. Non-limiting examples of a test sampleinclude a tissue specimen, a biopsy specimen, a surgical specimen, acervical swab, a cytological specimen, a plasma specimen, a serumspecimen, and a urine specimen. In some embodiments, the test samplefrom the woman is from a specimen selected from the group consisting ofa tissue specimen, a biopsy specimen, a surgical specimen, a cervicalswab, a cytological specimen, a plasma specimen and a urine specimen. Insome embodiments, the test sample from the woman comprises a specimenselected from the group consisting of liquid prep, plasma, serum, andurine. As used herein, the term “liquid prep sample” refers to a samplethat is in a fluid, such as a sample comprising cervical cells in apreservative liquid. Particularly, “liquid prep sample” can refer to asolution that comprises cervical cells that have been collected from awoman, such as by a tool (e.g., a brush, a self-collection swab, atampon, and the like). In some embodiments, the test sample refers tothe liquid prep sample where the Pap and HPV test were performed,otherwise known as a reflex test. In some embodiments, the test samplerefers to a self-collected cervical cytology or urine sample. Testsamples will desirably contain cervical cells when they are collectedfrom the cervical mucosa and/or circulating DNA from cervical cells,when it is isolated from urine as ccfDNA.

As used herein, a “nucleic acid” or “polynucleotide” refers to thephosphate ester polymeric form of ribonucleosides (adenosine, guanosine,uridine or cytidine; “RNA molecules”) or deoxyribonucleosides(deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; “DNAmolecules”), or any phosphoester analogs thereof, such asphosphorothioates and thioesters, in either single stranded form, or adouble-stranded helix. Double stranded DNA-DNA, DNA-RNA and RNA-RNAhelices are possible. The term nucleic acid molecule, and in particularDNA or RNA molecule, refers only to the primary and secondary structureof the molecule, and does not limit it to any particular tertiary forms.Thus, this term includes double-stranded DNA found, inter alia, inlinear or circular DNA molecules (e.g., restriction fragments),plasmids, and chromosomes. In discussing the structure of particulardouble-stranded DNA molecules, sequences may be described hereinaccording to the normal convention of giving only the sequence in the 5′to 3′ direction along the non-transcribed strand of DNA (i.e., thestrand having a sequence homologous to the mRNA).

It may be beneficial to extract nucleic acids from the cells prior totesting. Some techniques of testing may not require pre-extraction. Insome embodiments, as used herein, “obtaining” the nucleic acid from atest sample means using the nucleic acid in the test sample in an assay,such as a promoter methylation assay. In some embodiments, “obtaining”the nucleic acid means to separate the nucleic acid from other moleculesfound in the test sample, such as lipids, carbohydrates and proteins.Separating the nucleic acid may occur by any technique known in the art,for example, extraction with organic solvents, filtration,precipitation, absorption on solid matrices (e.g., silica resin,hydroxyapatite or ion exchange), affinity chromatography (e.g., viasequence specific capture or nucleic acid specific ligands), molecularexclusion chromatography, and the like. In some embodiments, the nucleicacid comprises DNA. In some embodiments, the nucleic acid comprisesccfDNA, which are relatively short, fragmented pieces of circulating DNAthat get filtered by the kidneys and can be isolated from urine. In someembodiments, the nucleic acid is from about 150 to about 250 base pairs.

In some embodiments, after obtaining nucleic acid from the test samplefrom a woman, a promoter methylation level of the promoter regions of agroup of genes is determined. It has been found that the promotermethylation level of the promoter regions of a group of genes exhibitincreased promoter methylation in women having CIN2+ lesions as comparedto women having no intraepithelial lesions or malignancy (NILM).

In some embodiments, the group of genes consists of, consistsessentially of, or comprises a combination of at least three of thewoman's endogenous genes. As used herein, the term “endogenous gene” ofa woman is a gene that originated within the woman, as compared to aviral gene found in a sample taken from the woman, which did notoriginate within the woman. In some embodiments, the group of genesconsists of, consists essentially of, or comprises ZNF516 (e.g., NCBIGene ID 9658), FKBP6 (e.g., NCBI Gene ID 8468), and INTS1 (e.g., NCBIGene ID 26173).

In some embodiments, the group of genes consists of, consistsessentially of, or comprises a combination of at least one of thewoman's endogenous genes and at least one HPV gene. In some embodiments,the combination of at least one of the woman's endogenous genes and atleast one HPV gene consists of, consists essentially of, or comprisesZNF516, FKBP6, and INTS1 and HPV gene HPV16-L1.

Any tests can be used to detect promoter methylation. Suitable testswhich can be used without limitation include lab-on-chip technology,microfluidic technologies, biomonitor technology, proton recognitiontechnologies (e.g., Ion Torrent), single cell third generationsequencing (e.g. PacBio or Oxford Nanopore MinION hand-held sequencer;http://phys.org/news/2015-06-professors-handheld-dna-sequencer.html),and other highly parallel and/or deep sequencing methods. In someembodiments, determining the promoter methylation level comprisesperforming bisulfite modification to the nucleic acid from the testsample to produce a bisulfite modified nucleic acid. DNA methylation isa biochemical process whereby a methyl group is added to the cytosine oradenine DNA nucleotides. CG dinucleotides tend to cluster in regionscalled CpG islands, mainly present in the promoters of genes. Promotermethylation can directly switch off gene expression by preventingtranscription factors binding to promoters.

Bisulfite compounds, for example, sodium bisulfite, convertnon-methylated cytosine residues to bisulfite modified cytosineresidues. The bisulfite ion treated gene sequence can be exposed toalkaline conditions, which convert bisulfite modified cytosine residuesto uracil residues. Sodium bisulfite reacts readily with the 5,6-doublebond of cytosine (but poorly with methylated cytosine) to form asulfonated cytosine reaction intermediate that is susceptible todeamination, giving rise to a sulfonated uracil. The sulfonate group canbe removed by exposure to alkaline conditions, resulting in theformation of uracil. The DNA can be amplified, for example, by PCR, andsequenced to determine whether CpG sites are methylated in the DNA ofthe sample. Uracil is recognized as a thymine by Taq polymerase and,upon PCR, the resultant product contains cytosine only at the positionwhere 5-methylcytosine was present in the starting template DNA. One cancompare the amount or distribution of uracil residues in the bisulfiteion treated gene sequence of the test cell with a similarly treatedcorresponding non-methylated gene sequence. A decrease in the amount ordistribution of uracil residues in the gene from the test cell indicatesmethylation of cytosine residues in CpG dinucleotides in the gene of thetest cell. The amount or distribution of uracil residues also can bedetected by contacting the bisulfite ion treated target gene sequence,following exposure to alkaline conditions, with an oligonucleotide thatselectively hybridizes to a nucleotide sequence of the target gene thateither contains uracil residues or that lacks uracil residues, but notboth, and detecting selective hybridization (or the absence thereof) ofthe oligonucleotide. Examples of performing bisulfite modification to anucleic acid to determine hypermethylation of the nucleic acid aredisclosed in U.S. Pat. No. 8,859,468, which is hereby incorporated byreference in its entirety. In some embodiments, the level of thebisulfite-modified nucleic acid is measured by sequencing of thebisulfite-modified nucleic acid.

In some embodiments, the level of the bisulfite modified nucleic acid ismeasured by quantitative real-time methylation specific PCR (QMSP). Ingeneral, PCR refers to an in vitro method for amplifying or replicatinga specific polynucleotide template sequence. The PCR reaction involves arepetitive series of temperature cycles. The reaction mix usuallycomprises dNTPs (each of the four deoxynucleotides dATP, dCTP, dGTP, anddTTP), primers, buffers, DNA polymerase, and target nucleic acidmolecule or template. The PCR step can use a variety of thermostableDNA-dependent DNA polymerases, such as Taq DNA polymerase, which has a5′-3′ nuclease activity but lacks a 3′-5′ proofreading endonucleaseactivity. In real-time or quantitative PCR, the DNA is amplified andsimultaneously quantified. QMSP protocols adopt the advantages ofreal-time PCR by using fluorescent-labeled MSP primers. Non-limitingexamples of QMSP variations include Syber green-based QMSP, sensitivemelting analysis after real-time MSP, and methylation-specificfluorescent amplicon generation.

In some embodiments, the quantitative real-time methylation specific PCR(QMSP) and/or sequencing of the bisulfite modified nucleic acid isperformed using primers and/or probes specific for the group of genes.In some embodiments, a combination of the woman's endogenous genesconsists of, consists essentially of, or comprises ZNF516, FKBP6, andINTS1 and HPV gene HPV16. In some embodiments, the primers and/or probesare selected from the group consisting of SEQ ID NOS: 1-12.

In some embodiments, the methylation status of the nucleic acid(s) ofthe one or more genes of interest are obtained by performing at leastone human DNA methylation detection assay selected from the groupconsisting of nucleic acid sequencing, PCR, a microarray assay, arestriction enzyme selection assay, a sequence capture assay, or anaffinity enrichment assay.

In some other embodiments, the methylation status of the nucleic acid isobtained by performing at least one HPV DNA methylation detection assayselected from the group consisting of nucleic acid sequencing, PCR, amicroarray assay, a restriction enzyme selection assay, a sequencecapture assay, or an affinity enrichment assay.

The terms “increased,” “increase,” “enhance,” or “activate” are all usedherein to generally mean an increase by a statically significant amount;for the avoidance of any doubt, the terms “increased,” “increase,”“enhance,” or “activate” means an increase of at least 10% as comparedto a reference level, for example an increase of at least about 20%, orat least about 30%, or at least about 40%, or at least about 50%, or atleast about 60%, or at least about 70%, or at least about 80%, or atleast about 90% or up to and including a 100% increase or any increasebetween 10-100% as compared to a reference level, or at least about a2-fold, or at least about a 3-fold, or at least about a 4-fold, or atleast about a 5-fold or at least about a 10-fold increase, or anyincrease between 2-fold and 10-fold or greater as compared to areference level.

The power of a diagnostic test to correctly predict status is commonlymeasured as the sensitivity of the assay, the specificity of the assayor the area under a receiver operated characteristic (“ROC”) curve.Sensitivity is the percentage of true positives that are predicted by atest to be positive, while specificity is the percentage of truenegatives that are predicted by a test to be negative. A ROC curveprovides the sensitivity of a test as a function of 1-specificity. Thegreater the area under the ROC curve, the more powerful the predictivevalue of the test. Other useful measures of the utility of a test arepositive predictive value and negative predictive value. Positivepredictive value is the percentage of people who test positive that areactually positive. Negative predictive value is the percentage of peoplewho test negative that are actually negative. Diagnostic tests that usethese biomarkers may show a ROC of at least about 0.6, at least about0.7, at least about 0.8, or at least about 0.9.

The promoter methylation level is differentially present in women havingCIN2+ lesions and women having no intraepithelial lesions or malignancy(NILM), and therefore, is useful in triaging a HPV-positive woman intocolposcopy. In certain aspects, the promoter methylation level of agroup of genes is measured in a test sample using the methods describedherein and compared, for example, to predefined promoter methylationlevels and correlated to Cervical Intraepithelial Neoplasia (CIN) grade.In particular aspects, the measurement(s) may then be compared with arelevant diagnostic amount(s), cut-off(s), or multivariate model scoresthat distinguish a NILM grade from a CIN2+ grade. The diagnosticamount(s) represents a measured amount of a promoter methylation levelof a group of genes above which or below which a woman is classified ashaving a particular CIN grade. As is well understood in the art, byadjusting the particular diagnostic cut-off(s) used in an assay, one canincrease the sensitivity or specificity of the diagnostic assay. Inparticular aspects, the diagnostic cut-off can be determined, forexample, by measuring the amount of promoter methylation levels of agroup of genes in a statistically significant number of samples fromwomen with the different CIN grades, and drawing the cut-off to suit thedesired levels of specificity and sensitivity.

In some embodiments, the presently disclosed method has a specificity ofat least 60%. In some embodiments, the method has a sensitivity of atleast 90%. In some embodiments, the method has a positive predictivevalue (PPV) of at least 52%. In some embodiments, the method has anegative predictive value (NPV) of at least 90%.

In some embodiments, the group of genes consists of, consistsessentially of, or comprises ZNF516, FKBP6, and INTS1 and thespecificity is at least 88%, the sensitivity is at least 88%, thepositive predictive value (PPV) is at least 93%, and the negativepredictive value (NPV) is at least 90%.

In some embodiments, the group of genes consists of, consistsessentially of, or comprises ZNF516, FKBP6, and INTS1 and HPV geneHPV16-L1, and the specificity is at least 60%, the sensitivity is atleast 90%, the positive predictive value (PPV) is at least 52%, and thenegative predictive value (NPV) is at least 93%.

In some embodiments, the presently disclosed methods further compriseenriching the nucleic acid from the test sample before determining thepromoter methylation level. In some embodiments, enriching the nucleicacid from the test sample comprises: (a) preparing a library of thenucleic acid from the test sample; (b) amplifying the library using PCRto form a pre-capture PCR library; (c) hybridizing the pre-capture PCRlibrary to a custom-designed pool of HPV-specific and human-specificcapture probes to form a post-capture PCR library; (d) amplifying thepost-capture PCR library to produce enriched nucleic acid; and (e)optionally repeating steps (c) and (d).

The term “enriching” as used herein means to purify or partially purifythe molecule of interest. In some embodiments, the custom-designed poolof HPV-specific and human-specific capture probes is designed to capturemost or all of the genomes of high-risk HPV-specific types. In someembodiments, the custom-designed pool of HPV-specific and human-specificcapture probes is designed to capture only some regions of thegenotype-specific regions of at least one high-risk HPV genome, such as1, 2, 3, 4, or 5 or more regions. In some embodiments, thecustom-designed pool of HPV-specific and human-specific capture probesis designed to capture 2 to 3 regions of the HPV genome thatdistinguishes high-risk from low-risk HPV types. In some embodiments,the custom-designed pool of HPV-specific and human-specific captureprobes does not capture low-risk HPV types. In some embodiments, thecustom-designed pool of HPV-specific and human-specific capture probesis designed to capture regions of the HPV16 genome. In some embodiments,the custom-designed pool of HPV-specific and human-specific captureprobes is designed to capture most or all of the genomes of the group ofgenes that exhibit increased promoter methylation in women having CIN2+lesions as compared to women having no intraepithelial lesions ormalignancy (NILM). In some embodiments, the custom-designed pool ofHPV-specific and human-specific capture probes is designed to capturemost or all of the promoters of the group of genes that exhibitincreased promoter methylation in women having CIN2+ lesions as comparedto women having no intraepithelial lesions or malignancy (NILM). In someembodiments, the custom-designed pool of HPV-specific and human-specificcapture probes is designed to capture most or all of the promoters ofZNF516, FKBP6, and INTS1.

In some embodiments, the presently disclosed subject matter provides amethod for triaging a human papillomavirus (HPV)-positive woman intocolposcopy, the method comprising: (a) selecting a HPV-positive womantesting positive for one or more high risk types of HPV; (b) providing akit that comprises the probes and/or primers needed for determining inthe nucleic acid from a test sample of the selected HPV-positive woman apromoter methylation level of the promoter regions of a group of genesthat exhibit increased promoter methylation in women having CIN2+lesions as compared to women having no intraepithelial lesions ormalignancy (NILM); (c) obtaining nucleic acid from the test sample fromthe selected HPV-positive woman; (d) determining the promotermethylation level in the nucleic acid from the test sample of theselected HPV-positive woman using the provided kit; and (e) triaging theHPV-positive woman into colposcopy when the level of promotermethylation of the group of genes is increased relative to the level ofpromoter methylation of the group of genes in a reference sampleobtained from women having NILM. In some embodiments, the kit comprisesthe probes and/or primers needed for determining in the nucleic acidfrom the test sample of the selected HPV-positive woman the promotermethylation level of the promoter regions of the group of genesconsisting of, consisting essentially of, or comprising ZNF516, FKBP6,and INTS1 and HPV gene HPV16-L1.

In some embodiments, the presently disclosed subject matter furthercomprises informing the woman or a treating physician of the result ofthe method of triaging the human papillomavirus (HPV)-positive womaninto colposcopy. In some embodiments, the presently disclosed methodsfurther comprise performing a colposcopy. In some embodiments, thepresently disclosed subject matter further comprises providing aprognosis regarding the development of cervical cancer based on thecolposcopy performed. In some embodiments, for example if the presentlydisclosed methods are negative for promoter methylation, the methodsfurther comprise recommending that the woman continue to be screened orretested at regular intervals, such as every six months, every year,every two years, every three years or more.

In some embodiments, the presently disclosed subject matter furthercomprises diagnosing the woman as having cervical pre-cancerous lesionsand/or cervical cancer.

In some embodiments, the presently disclosed subject matter furthercomprises recommending treatment and/or treating the woman. Treatmentmay include removal of precancerous lesions; radiation treatment;surgery, such as a hysterectomy, removal of lymph nodes, a cone biopsy,and/or a trachelectomy; and/or chemotherapy, using for example aplatinum-containing anti-cancer drug (e.g. cisplatin) and/or atopisomerase inhibitor (e.g., topotecan). In some embodiments, thetreatment is selected from the group consisting of removal ofprecancerous lesions, radiation treatment, surgery, and chemotherapy.

In some embodiments, the presently disclosed methods further comprisemonitoring the efficacy of the treatment. Monitoring may occur byperforming a Pap smear, performing a colposcopy, testing for thepresence of a high risk type of HPV, and/or using the presentlydisclosed methods. In some embodiments, monitoring the efficacy of thetreatment occurs by at least one method selected from the groupconsisting of performing a Pap smear, performing a colposcopy, testingfor the presence of a high risk type of HPV, and determining a promotermethylation level of the promoter regions of a group of genes thatexhibit increased promoter methylation in women having CIN2+ lesions ascompared to women having no intraepithelial lesions or malignancy(NILM).

II. Kits for Triaging a Human Papillomavirus (HPV)-Positive Woman intoColposcopy

The presently disclosed subject matter also relates to kits forpracticing the methods of the invention. By “kit” is intended anyarticle of manufacture (e.g., a package or a container) comprisingprimers and/or probes specific for a group of genes that exhibitincreased promoter methylation in women having CIN2+ lesions as comparedto women having no intraepithelial lesions or malignancy (NILM).

In some embodiments, the presently disclosed subject matter provides akit for triaging a human papillomavirus (HPV)-positive woman intocolposcopy, the kit comprising: (a) a container containing primersand/or probes specific for a group of genes that exhibit increasedpromoter methylation in women having CIN2+ lesions as compared to womenhaving no intraepithelial lesions or malignancy (NILM); and (b)instructions for use of the primers and/or probes in triaging a humanpapillomavirus (HPV)-positive woman into colposcopy.

In some embodiments, the group of genes consists of, consistsessentially of, or comprises ZNF516, FKBP6, and INTS1. In someembodiments, the group of genes consists of, consists essentially of, orcomprises ZNF516, FKBP6, and INTS1 and HPV gene HPV16. In someembodiments, the primers and/or probes are selected from the groupconsisting of SEQ ID NOS: 1-12.

III. Methods for Treating Cervical Cancer

In some embodiments, the presently disclosed subject matter providesmethods for treating cervical cancer. In some embodiments, the presentlydisclosed subject matter provides a method for treating cervical cancer,the method comprising: (a) selecting a HPV-positive woman testingpositive for one or more high risk types of HPV; (b) providing a kitthat comprises the probes and/or primers needed for determining in thenucleic acid from a test sample of the selected HPV-positive woman apromoter methylation level of the promoter regions of a group of genesthat exhibit increased promoter methylation in women having CIN2+lesions as compared to women having no intraepithelial lesions ormalignancy (NILM); (c) obtaining nucleic acid from the test sample fromthe selected HPV-positive woman; (d) determining the promotermethylation level in the nucleic acid from the test sample of theselected HPV-positive woman using the provided kit; (e) triaging theHPV-positive woman into colposcopy when the level of promotermethylation of the group of genes is increased relative to the level ofpromoter methylation of the group of genes in a reference sampleobtained from women having NILM; (f) performing a colposcopy; (g)diagnosing the woman as having cervical pre-cancerous lesions and/orcervical cancer; and (h) treating the woman for cervical cancer.Optionally, the presently disclosed methods include providing aprognosis regarding the development of cervical cancer based on thecolposcopy performed.

Treatment of the woman for cervical cancer may include removal ofprecancerous lesions; radiation treatment; surgery, such as ahysterectomy, removal of lymph nodes, a cone biopsy, and/or atrachelectomy; and/or chemotherapy. Non-limiting therapeutic agents thatcan be used in the treatment of the woman for cervical cancer includeplatinum-containing anti-cancer drug (e.g., cisplatin, carboplatin),topisomerase inhibitors (e.g., topotecan), tubulin targeting agents(e.g., paclitaxel), pyrimidine analogs (e.g, fluorouracil), nucleosideanalogs (e.g., gemcitabine), anti-mitotic agents (e.g., docetaxel), andalkylating agents (e.g., cyclophosphamide).

Following long-standing patent law convention, the terms “a,” “an,” and“the” refer to “one or more” when used in this application, includingthe claims. Thus, for example, reference to “a subject” includes aplurality of subjects, unless the context clearly is to the contrary(e.g., a plurality of subjects), and so forth.

Throughout this specification and the claims, the terms “comprise,”“comprises,” and “comprising” are used in a non-exclusive sense, exceptwhere the context requires otherwise. Likewise, the term “include” andits grammatical variants are intended to be non-limiting, such thatrecitation of items in a list is not to the exclusion of other likeitems that can be substituted or added to the listed items.

For the purposes of this specification and appended claims, unlessotherwise indicated, all numbers expressing amounts, sizes, dimensions,proportions, shapes, formulations, parameters, percentages, parameters,quantities, characteristics, and other numerical values used in thespecification and claims, are to be understood as being modified in allinstances by the term “about” even though the term “about” may notexpressly appear with the value, amount or range. Accordingly, unlessindicated to the contrary, the numerical parameters set forth in thefollowing specification and attached claims are not and need not beexact, but may be approximate and/or larger or smaller as desired,reflecting tolerances, conversion factors, rounding off, measurementerror and the like, and other factors known to those of skill in the artdepending on the desired properties sought to be obtained by thepresently disclosed subject matter. For example, the term “about,” whenreferring to a value can be meant to encompass variations of, in someembodiments, ±100% in some embodiments ±50%, in some embodiments ±20%,in some embodiments ±10%, in some embodiments ±5%, in some embodiments±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from thespecified amount, as such variations are appropriate to perform thedisclosed methods or employ the disclosed compositions.

Further, the term “about” when used in connection with one or morenumbers or numerical ranges, should be understood to refer to all suchnumbers, including all numbers in a range and modifies that range byextending the boundaries above and below the numerical values set forth.The recitation of numerical ranges by endpoints includes all numbers,e.g., whole integers, including fractions thereof, subsumed within thatrange (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5,as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like)and any range within that range.

EXAMPLES

The following Examples have been included to provide guidance to one ofordinary skill in the art for practicing representative embodiments ofthe presently disclosed subject matter. In light of the presentdisclosure and the general level of skill in the art, those of skill canappreciate that the following Examples are intended to be exemplary onlyand that numerous changes, modifications, and alterations can beemployed without departing from the scope of the presently disclosedsubject matter. The synthetic descriptions and specific examples thatfollow are only intended for the purposes of illustration, and are notto be construed as limiting in any manner to make compounds of thedisclosure by other methods.

Example 1

Human Papilloma Virus (HPV) testing is increasingly used for cervicalcancer screening in conjunction with cervical cytology. Althoughprivacy, cultural, and infrastructure issues challenge the effectiveimplementation of HPV testing for cervical cancer screening worldwide,several countries have already implemented HPV testing in theirscreening protocols. There are currently no tests that can reliablyidentify the patients with abnormal cytology and positive oncogenic HPVresults (HPV+) that need to be referred for colposcopy. A triage testfrom cytology to colposcopy needs to be able to discriminate in thecytology sample those patients with a Cervical Intraepithelial Neoplasia(CIN) grade more likely to progress to cervical cancer (CIN2+).

We set out to identify a panel of methylated human papilloma virus (HPV)and human genes that can discriminate between CIN2+ and normal/CIN1patients in liquid prep samples and transrenal DNA isolated from urine(ccfDNA).

Methods: We used three independent cohorts, from Chile, Baltimore, andPuerto Rico, to develop a method that can be used to triage intocolposcopy, HPV+ women with abnormal cytology. Participants were womenwith no cervical intraepithelial lesions or malignancy, and women with:abnormal cervical biopsies (CIN1, CIN2, CIN3); carcinoma in-situ, andcervical cancer.

Using DNA methylation arrays for Discovery and quantitative MethylationSpecific PCR (qMSP) for Validation, we found that promoter methylationof ZNF516, FKBP6, and INTS1 discriminates samples with CIN2+ lesionsfrom samples with no intraepithelial lesions or malignancy (NILM): 88.3%sensitivity, 88.9% specificity, 93.2 Area Under the Curve (AUC), 86.9%positive predictive value (PPV) and 90.2% negative predictive value(NPV). Using custom sequence capture pools of baits, we pulled downgenomic and bisulfate converted high-risk HPV DNA before library prepfor NGS in 454 and MiSeq instruments, respectively. Using our NGSresults, we optimized a Syber Greeen qPCR assay to detect high risk HPVDNA and a qMSP primer-probe set to detect methylated HPV. We replicatedthe results in liquid prep samples (n=67), adding HPV16 methylation tothe panel: 90.9% sensitivity, 60.9% specificity, 90.1 (AUC), 52.6%positive predictive value (PPV) and 93.3% NPV. These results wereverified in plasma DNA (AUC=80.7) and ccfDNA (AUC=86.1) isolated from asubset of patients who provided the liquid prep samples.

Our results suggest that our panel of viral and host gene methylationmarkers may be used as a reflex test in liquid prep to triage high riskHPV positive women into colposcopy, or as a screening and triage test inccfDNA, in combination with our high risk HPV test. The presentlydisclosed methods can be used to triage into colposcopy, women withabnormal Pap smears and positive oncogenic HPV results (HPV+) using hostand viral DNA methylation markers in liquid prep and ccfDNA.

Results

DNA samples isolated from cervical brush (n=211), liquid cytology(n=107), serum (n=40) and ccfDNA (n=130) from women with normal cervicalepithelium, premalignant cervical neoplasia lesions and cervical cancerlesions were examined to test the performance of viral and host DNAmethylation markers as classifiers for triage into colposcopy (FIGS.1a-b ). We first identified a panel of host DNA methylation markersassociated with CIN2+ biopsy lesions using genome-wide DNA methylationarrays for Discovery and qMSP for Validation in cervical brush samplesfrom Chile. This classifier was previously associated with cervicalcancer and abnormal cytology samples in the same cohort (23).

Cervical brush samples (n=211) were genotyped for HPV with the ReverseLine Blot assay. After removing samples without a definitive biopsyresult, we examined the promoter methylation frequency of three genes(ZNF516, INTS1 and FKBP6) in cervical brush samples from women withnormal (n=34), CIN1 (n=34), CIN2 (n=33) and CIN3 (n=20) and cervicalcancer (n=90) pathology reports. We compared samples from women with nointraepithelial lesions or malignancy (NILM) and CIN1 lesions withsamples from women with CIN2+ lesions, and found that: ZNF516 has 91.7%Sensitivity, 27.4% Specificity and an AUC of 0.62; FKBP6 has 92.4%Sensitivity, 46.8% Specificity and an AUC of 0.68; INTS1 has 93.8%Sensitivity, 30.3% Specificity and an AUC of 0.57. We then comparedsamples from women with NILM to samples from women with CIN2+ lesions,we found that: ZNF516 has 91.7% Sensitivity, 38.9% Specificity and anAUC of 0.76; FKBP6 has 90.9% Sensitivity, 67.6% Specificity and an AUCof 0.86; INTS1 has 90.7% Sensitivity, 40.5% Specificity and an AUC of0.62.

The panel of three classifiers (ZNF516, INTS1 and FKBP6) has 90%Sensitivity, 88.9% Specificity, an AUC of 0.93, a Positive PredictiveValue (PPV) of 93.1% and a Negative Predictive Value (NPV) of 84.2%,when comparing women with no intraepithelial lesions or malignancy(NILM) with women with CIN2+ lesions (Table 1 below, FIGS. 2a and 2b ).

TABLE 1 Table 1. Promoter methylation frequency in cervical brushsamples Chile - cytobrush n = 211 Lesion Sensitivity Specificity AUC PPVNPV comparison Marker % % % % % CIN2+ vs. ZNF516 91.7 27.4 61.6NILM/CIN1 FKBP6 92.4 46.8 68.4 INTS1 93.8 30.3 56.9 3 gene panel 60 74.577 57.1 76.7 CIN2+ vs. ZNF516 91.7 38.9 76.5 NILM FKBP6 90.9 67.6 85.6INTS1 90.7 40.5 62.2 3 gene panel 88.3 88.9 93.2 86.9 90.2

We then tested this panel of three classifiers in liquid-based cytologysamples (n=67) from women in Puerto Rico, a subset of which had beenpreviously tested for concordance of high risk HPV genotype betweenliquid prep and urine DNA (30). We compared samples from women with NILMand CIN1 lesions with samples from women with CIN2+ lesions and foundthat: ZNF516 has 72.7% Sensitivity, 48.1% Specificity and an AUC of0.63; FKBP6 has 63.6% Sensitivity, 34.6% Specificity and an AUC of 0.50;INTS1 has 91% Sensitivity, 35% Specificity and an AUC of 0.66. We alsotested the performance of HPV16-L1 methylation, using previouslydesigned primers and probes (39), and found it had 63.6% Sensitivity,57.7% Specificity and an AUC of 0.54. Then we compared samples fromwomen with NILM with samples from women with CIN2+ lesions, we foundthat: ZNF516 has 63.6% Sensitivity, 17.4% Specificity and an AUC of0.50; FKBP6 has 63.6% Sensitivity, 39.1% Specificity and an AUC of 0.50;INTS1 has 63.6% Sensitivity, 39.1% Specificity and an AUC of 0.47. Wealso tested the performance of HPV16-L1 methylation and found it had63.6% Sensitivity, 100% Specificity and an AUC of 0.79.

The panel of four classifiers in liquid prep samples, ZNF516, INTS1,FKBP6 and HPV16-L1 has 90.9% Sensitivity, 60.9% Specificity, an AUC of0.90, a PPV of 52.6% and a NPV of 93.3% when comparing NILM with CIN2+lesions (Table 2 below, FIGS. 3A and 3B).

TABLE 2 Table 2. Promoter methylation frequency in liquid prep samplesUPR - Liquid prep n = 67 Lesion Sensitivity Specificity AUC PPV NPVcomparison Marker % % % % % CIN2+ vs. ZNF516 72.7 48.1 63.5 NILM/CIN1FKBP6 63.6 34.6 49.8 INTS1 90.9 34.6 66.1 HPV16-L1 63.6 57.7 54.2 4 genepanel 90.9 46.15 73.1 26.3 96 CIN2+ vs. ZNF516 63.6 17.4 50 NILM FKBP663.6 39.1 50.1 INTS1 63.6 39.1 46.6 HPV16-L1 63.6 100 78.7 4 gene panel90.9 60.9 90.1 52.6 93.3

Example 2

Sequencing the hrHPV Genome in Urine ccfDNA

To enable the testing of this four gene panel in ccfDNA, we optimized apreviously published ccfDNA isolation method and compared it to the goldstandard, phenol chloroform DNA extraction method (FIG. 13). Wedeveloped sequence capture methods and quantitative PCR assays tomeasure HPV high-risk types and methylated HPV CpGs in ccfDNA from womenwith and without cervical dysplasia.

To examine the hrHPV genome in urine ccfDNA we developed customdual-sequence capture baits (FIG. 7), to enrich samples for hrHPV DNAfrom 12 high-risk types (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58,and 59). To assess the enrichment efficiency of the dual sequencecapture method we developed the hrHPV ccfDNA-qPCR assay with primersthat amplify the HPV E1 region common to 13 high-risk HPV types (HPV 16,18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68). We comparedpre-capture and post-capture DNA from cervical cancer cell lines thatharbor high risk HPV, HeLa (HPV18) and CSCC7 (HPV16). PicoGreenquantitation was performed on both pre-capture and post-capture DNAsamples, and a normalized amount of DNA (5 ng) was analyzed. FIG. 8shows an amplification plot demonstrating the successful amplificationand enrichment obtained with the dual sequence capture assay. Delta Ctvalues for HeLa and CSCC7 were 14.88 and 12.66, respectively. Based onan estimated efficiency for the assay, approximate fold enrichment wasgreater than 1700.

To assess the enrichment efficiency of the dual sequence capture methodon clinical samples, we compared the HPV ccfDNA-qPCR assay resultsobtained on eight (8) pre-capture and post-capture HPV-enrichedcirculating DNA samples obtained from patients with CIN1 (n=4) andCIN2-3 (n=4). As shown in FIG. 9, with data representing five clinicalsamples, the average Delta Ct value for Pre-Capture vs. Post-CaptureHPV-enriched ccfDNAs was 11.07, for an average fold enrichment of 670 ofHPV ccfDNA in patient samples.

We then examined if the HPV ccfDNA-qPCR assay could discriminate ccfDNAisolated from women with and without cervical dysplasia. FIG. 4 showsthe amplification curves for the HPV ccfDNA-qPCR assay on ccfDNA fromCIN2-3 (n=14), CIN1 (n=13) and 10 samples from women with NILM. Thefrequency of dysplastic samples that amplified differed significantlyfrom NILM samples, but not with lesion severity: NILM (30%), CIN1 (77%,p=0.02), and CIN2+ (71%, p=0.04).

We then used massively parallel next generation sequencing to quantifythe different HPV genotypes present in ccfDNA from 7 patients with CIN1(n=3) and CIN2-3 (n=4), using DNA from two cervical cancer cell lines(HeLa and CSCC7) as positive controls. The DNA was enriched for highrisk HPV DNA with the custom dual sequence capture high risk HPV assayprior to multiplexed sequencing on a 454 GS Junior (Roche) system. Themultiplexed massively parallel sequencing runs produced 230,385 readswith an average length of 138.3 bp. The reads in the clinical samplescovered 82-100% of the reference HPV 16 and 73-100% of the HPV 18genomes, with an average of 21% (HPV18) to 33% (HPV18) percentage of allreads mapping to the sequences. These results were comparable to thepercentage of all reads that mapped to the reference sequences in thepositive control samples: 92% of the CSCC7 reads mapped on targetcovering 77% of the HPV16 genome; and 89% of the HeLa reads mapped to67% of HPV 18, as expected (Table 3).

TABLE 3 Table 3. Dual sequence capture of high risk HPV in TrDNA DualSequence capture of high risk HPV in TrDNA aligned to HPV 16 and HPV 18HPV16ref HPV18ref Input unique % of unique % of DNA # match all % matchall % Sample ID Lesion HPV (ng) Reads reads reads coverage reads readscoverage HeLa cancer 18 250 51952 cell line with hpv18 46284 89.1 67.07CSCC7 cancer 16 250 48095 44262 92 76.6 cell line with hpv16 TrDNA445CIN3 16.45 60 25998 24670 95 100 772 3 73.4 TrDNA455 CIN1 18 100 55002144 0.3 81.6 53075 96.5 100 TrDNA456 CIN1 33 100 45982 953 2.1 91.4 16473.6 74.3 TrDNA481 CIN3 16 55 18159 6700 37 100 1185 6.5 87.1 TrDNA504CIN3 16 60 33218 11227 33.8 99.4 4624 13.9 100 TrDNA513 CIN1 16.66 3025242 5085 20.1 100 2202 8.7 100 TrDNA571 CIN3 16 100 26472 12229 46 1003989 15.1 100 Average 72 32868 8715 33 96 9635 21 91

To quantify the presence and HPV genotype composition of the patientsamples, we first aligned the reads against the >170 human HPV genomesin the PapillomaVirus Episteme (PaVE) database, maintained by the NIHNIAID Samples TrDNA_445 and TrDNA_455 had more than 80% of their readsmapped to HPV 16 and HPV 18, however, the remaining five samples weresomewhat split, mapping to both HPV 16 and 18 genotypes, with many notmapping to either (FIG. 14 and Table 4 below).

TABLE 4 Profiling of HPV TrDNA 454 reads by tiered mapping READS Humanpapillomavirus type 193 Human papillomavirus type 44, complete genome 24Human papillomavirus type 61, complete genome 841 Human papillomavirustype 56 clone Qv24970, complete genome 243 Human papillomavirus type 45isolate Qv31035, complete genome 231 Human papillomavirus type 72 109Human papillomavirus type 11 isolate LZod45-11; complete genome 837Human papillomavirus type 42 isolate TJ43-42, complete genome 54 Humanpapillomavirus type 53 isolate TJ43-53, complete genome 493 Humanpapillomavirus type 18 complete sequence 732 Human papillomavirus type16, complete genome 421 Human papillomavirus type 68b, complete genome226 Human papillomavirus type 6 complete genome, isolate CAC231 156Human papillomavirus 39 330 Human papillomavirus type 31 isolateQV12357, complete genome 10060 Human papillomavirus type 33 isolateQv34189, complete genome 564 Human papillomavirus type 35 isolateQV29782, complete genome 145 Human papillomavirus type 52 isolateQv-03594, complete genome 1533 Human papillomavirus type 58 isolateZ094, complete genome 184 Human papillomavirus type 81 complete genome17645 Human papilloma virus type 59, complete viral genome 48 Humanpapillomavirus 54, complete genome 10913 Unmapped

To resolve the remaining reads, we searched the human genome using theprogram Bowtie (39), and then a local copy of the database of NCBIreference bacterial genomes, using BLAST (40). Following this tieredmapping approach, only a small number of reads were still mapping tounknown genomes, a small number of which (<50) were linker contaminants,while the others could potentially represent novel HPV genotypes orother viruses or bacteria (FIG. 14 and Table 4 above, representingsample TrDNA_456). FIGS. 4, 8 and 9 show the percentage of reads thatalign to different HPV types, human, bacteria and unknown genomes forthis same sample, TrDNA_456. Table 4 lists the reads mapping totwenty-one (21) HPV types after profiling reads of the clinical sampleTrDNA-456 by tiered read mapping. FIG. 10 shows the percentage of readsthat map to thirteen (13) HPV types, human, bacteria and unknown genomesfor clinical sample TrDNA-456.

Cloud-Based Visualization Tools of the HPV Genome for PersonalizedMedicine

We used two Cloud-based tools, which allow the comparative visualizationof HPV genomes uploaded by users against a reference genome of interest(43). Our demonstration cloud-based servers show the alignment resultsof 11 hrHPV types, 9 low risk HPV types, and seven clinical samples(TrDNA-445, TrDNA-455, TrDNA-456, TrDNA-481, TrDNA-504, TrDNA-513, andTrDNA-571) against a reference genome (HPV16).

The large-scale HPV DNA server(http://enterix.cbcb.umd.edu/enteric/enteric-hpv.html) produces agraphical ‘large-scale’ view of the pairwise alignments of twenty HPVgenomes against a reference genome (HPV16 in this demo), together withannotations of genome rearrangement events (FIG. 14 top).

The “close-up” HPV DNA server(http://enterix.cbcb.umd.edu/menteric/enteric-hpv.html) computes anddisplays nucleotide-level (close-up′) multiple alignments of sequencesin a 1 Kb region starting at a user-specified address or gene in thereference genome (FIG. 14 bottom).

Sequencing the hrHPV Epigenome in Urine ccfDNA

To examine the hrHPV genome in urine ccfDNA we used the Sure SelectMethyl-Seq Target Enrichment (Agilent) assay to enrich DNA samples fromtwo HPV16 positive cervical cancer cell lines, CaSki (ATCC® CRL-1550™,600 integrated HPV16 copies) and SiHa (ATCC® HTB35™, 2 integrated HPV16copies); two HPV16 positive Head and Neck Squamous Cell Carcinoma celllines, SCC-47 and SCC-90; and urine ccfDNA from two clinical samples:one from a patient with ASCUS and CIN1 (TrDNA47) and another one from apatient with HSIL and CIN3 (TrDNA50). Samples were PCR-amplified usingsample-specific indexed (“barcoding”) primers for multiplexed sequencingon a MiSeq (Illumina) system.

Reads from the FASTQ files were aligned to all HPV types in the PAVEdatabase and we performed CpG methylation analysis using Bismark,modified to analyze high-risk HPV genomes. The multiplexed massivelyparallel sequencing run produced 14,442,406 reads with a length of 100bp. The percentage of all reads of the HPV positive cervical cancer andhead and neck cancer squamous cell carcinoma (HNSCC) cell lines thatmapped uniquely to some of the reference genomes in the PAVE databasecan be seen in the top panel of (Table 5 below) Caski (93%); SiHa (13%);SCC-047 (68%) and SCC-090 (87%).

TABLE 5 Alignment of HPV genotype from the custom sequence capturemethod against the PapillomaVirus Episteme (PaVE) database SampleReference_genome Total_Reads Reads_No_Alignment Reads_Aligned_uniquelyMapping_efficiency Caski HPV_pave_all 1648170 119552 1528618 93% SiHaHPV_pave_all 1838822 1597909 240913 13% SCC-047 HPV_pave_all 2391854759528 1632326 68% SCC-090 HPV_pave_all 2806373 363874 2442499 87%TrDNA-34(CIN1) HPV_pave_all 3183371 3181520 1848 0% TrDNA-50(CIN3)HPV_pave_all 2573816 2568941 4869 0% Total 14442406 8591324 5851073Average 2407068 1431887 975179 44% Caski HPV-16 142622 1505548 91% SiHaHPV-16 1602013 236809 13% SCC-047 HPV-16 814051 1577803 66% SCC-090HPV-16 397578 2406795 86% TrDNA-34(CIN1) HPV-16 3181642 1729 >1%TrDNA-50(CIN3) HPV-16 2572738 1078 >1% Total 8712644 5729762 Average1452107 954960 43% Caski HPV_16_11 1356064 292106 18% SiHa HPV_16_111791950 46872 3% SCC-047 HPV_16_11 2043910 347944 15% SCC-090 HPV_16_112375812 430561 15% TrDNA-34(CIN1) HPV_16_11 3182952 419 >1%TrDNA-50(CIN3) HPV_16_11 2573606 210 >1% Total 13324294 1118112 Average2220716 186352 9%Towards Personalized HPV Methylation Landscapes

The percentage of methylation across the HPV genome in all six sampleswas obtained with the Methylator Extractor module in Bismark.Scatterplots of the percentage of methylation by chromosomal location inthe HPV genome for each of the six samples are show in FIG. 4. Thepercentage of methylation is shown on the Y-axis of the upper panel. TheX-axis shows the chromosomal location along the HPV genome for bothpanels, including the promoters at positions 97 and 670 of the HPVgenome. The upper panel of each plot represents the percentage of CpGmethylation. The bottom panel of each plot represents the HPV genes andthe Upper Regulatory Region (41).

The different HPV genomes that aligned to the six samples are shown indifferent colors and shapes (HPV16-black dots; HPV35 red squares; HPV52red triangles; and HPV71 green squares). The two cervical cancer celllines had different patterns of CpG methylation. Caski had overallhigher levels of methylation than SiHA for CpGs across the genome. SiHAexhibits a bimodal distribution of methylation percentage. The majorityof CpGs below the 3500 position in the HPV genome have less than 60%methylation, while CpGs located between 3500 and 7200 positions on theHPV genome show over 80% methylation. This may be related to the lownumber of HPV copies per genome present in this cell line. Themethylation patterns for both HPV positive HNSCC cell lines were verysimilar to those observed in Caski. The clinical samples aligned to morethan one HPV type. TrDNA-34, a sample obtained from a patient with ASCUSand CIN1, aligned to HPV16 and HPV35. TrDNA-50, a sample obtained from apatient with HSIL and CIN3, aligned to HPV16, HPV52 and HPV71. Themethylation patterns of HPV16 in both clinical samples are very similarto the methylation patterns observed in Caski and both HNSCC cell lines,albeit with less abundant number of reads, as expected. Methylation ofthe remainder of HPV types was low overall.

To examine the HPV16 CpG methylation patterns we aligned the reads fromthe four cell lines to the HPV16 reference genome. The mappingefficiency (the percentage of total reads that aligned uniquely to thereference genome) to the HPV16 reference genome was very similar to thepercentage of all reads that mapped to the PAVE reference database forthe four cell lines: Caski (91%); SiHa (13%); SCC-047 (66%), and SCC-090(86%). The mapping efficiency of the reads from the HNSCC cell lines tothe HPV16 reference genome was in the range between the mappingefficiency obtained with SiHA and Caski, namely 86% for SCC-90 and 66%for SCC-47 (Table 5, middle panel). Since we know the number of copiesof HPV16 DNA in SiHa (2) and Caski (˜600), these sequencing results maybe a good indicator of the number of HPV copies present in the HNSCCcell lines.

The clinical samples aligned to more than one HPV type. TrDNA-34, asample obtained from a patient with ASCUS and CIN1, aligned to HPV16 andHPV35. TrDNA-50, a sample obtained from a patient with HSIL and CIN3,aligned to HPV16, HPV52 and HPV71. The methylation patterns of HPV16 inboth clinical samples are very similar to the methylation patternsobserved in Caski and both HNSCC cell lines, albeit with less abundantnumber of reads, as expected. Methylation of the remainder of HPV typeswas low overall.

Given that HPV type is defined by a measure of sequence divergence inthe HPV L1 region of the genome, we divided the reads from the four celllines that mapped only to the HPV16 L1 gene by the reads of the fourcell lines that mapped to the HPV16 genome, to calculate their mappingefficiency to the HPV16-L1 gene. The mapping efficiency of the four celllines to the HPV-16 L1 gene was high: Caski (81%); SiHA (80%); SCC-47(78%) and SCC-90 (82%). Surprisingly, the mapping efficiency of theclinical samples to the HPV16 L1 gene was as high as for the positivecontrols: 76% for TrDNA-34 and 81% for TrDNA-50 (Table 6).

TABLE 6 Mapping efficiency to HPVL1 Mapping_Efficiency Samples to_HPVL1Caski 81% SCC-047 78% SCC-090 82% SiHa 80% TrDNA-34 76% TrDNA-50 81%

We wanted to assess whether methylation levels in the HPV16-L1 variableregion could be used as a marker of progression in cervical cancerpremalignant lesions. To determine whether the L1 region is uniformlyrepresented in the reads, we calculated the ratio of the reads from eachof the four cell lines that mapped only to the HPV16 L1 gene (Table 5,bottom panel) to the total number of reads from each cell line thatmapped to the HPV16 genome, and determined the mapping efficiency to theHPV16-L1 gene. The mapping efficiency of the four cell lines to theHPV-16 L1 gene was high: Caski (81%); SiHA (80%); SCC-47 (78%) andSCC-90 (82%). The mapping efficiency of the clinical samples to theHPV16 L1 gene was as high as for the positive controls: 76% for TrDNA34and 81% for TrDNA50 (FIG. 11).

To further determine if HPV16-L1 methylation levels can be used as asurrogate marker of methylation of the HPV16 genome in urine ccfDNA, weexamined the distribution of CpG methylation after aligning the urineccfDNA samples to the HPV16-L1 gene. The CpG methylation median in theclinical samples is significantly higher than in the cell lines andhigher in urine ccfDNA from the CIN3 than from the CIN1 clinical sample(p<0.05), as expected (FIG. 11).

Quantification of Viral and Host DNA Methylation in Plasma and UrineccfDNA

To enable the testing of this four-gene panel in urine ccfDNA, weoptimized a previously published urine ccfDNA isolation method andcompared it to the gold standard, phenol chloroform DNA extractionmethod (FIG. 13, Top). We then designed primers and Taqman probes toquantify HPV16 L1 DNA methylation and ZNF516, INTS1 and FKBP6methylation in fragmented urine ccfDNA using qMSP. The genomic region ofthe HPV16 L1 gene used to design the Primers were designed to amplify inurine ccfDNA short amplicons (80 base-pairs long) of the same genomicregions previously used to quantify HPV16-L1 methylation in HNSCC (39);and ZNF516, INTS1 and FKBP6 in cervical cancer (18),

In a feasibility study we found that HPV16-L1 qMSP methylation candiscriminate bisulfite treated urine ccfDNA from patients with normalcytology (n=10) from women with dysplastic cytology and premalignantcervical lesions (ASCUS n=8; CIN1 n=3; CIN2+ n=3) with 100% sensitivityand specificity (FIG. 12).

We then quantified the methylation levels of the panel of viral and hostDNA genes in plasma and urine ccfDNA samples from women with NILM andCIN2+ lesions. In plasma, we found the panel of four classifiers has85.7% Sensitivity, 60.9% Specificity, an AUC of 0.807, PPV of 40% and aNPV of 93.3% (FIG. 6a ). In urine ccfDNA, we found the panel of fourclassifiers has 75% Sensitivity, 83.3% Specificity, an AUC of 0.861, PPVof 50% and a NPV of 93.8% (FIG. 6b ).

Discussion

The present inventors set out to identify a panel of methylated HPV andhuman host genes that can discriminate between CIN2+ and normal/CIN1lesions in a reflex test performed in liquid prep samples and ccfDNA inplasma and urine. The inventors are the first to show that a panel ofhost and viral DNA methylation markers can discriminate between CIN2+and NILM in multiple body compartments from the same individual: liquidprep, serum and urine. The results show that a precision medicine panelcan be used as a reflex test in liquid prep to triage women referred tocolposcopy. NGS reads from urine ccfDNA can be aligned in customcloud-based servers for life-course personalized cervical cancerscreening.

Women with low probability of having a CIN2+ lesion can be triaged outof a biopsy after colposcopy. The four-gene classifier best performed inliquid-prep with Sensitivity (90.9%), Specificity of 60.9% and NPV(93.3%). In urine ccfDNA, the four-gene classifier had equal NPV(93.8%), a better Specificity (83.3%) and similar AUC (0.861). Theresults obtained for this classifier in liquid prep and urine ccfDNAwarrant further study of this panel as a molecular biomarker to triagewomen referred to colposcopy after testing positive for high-risk HPVand being diagnosed with cervical dysplasia with cytology. Women withlow methylation values in this panel would be asked to return forfollow-up cytology and HPV co-testing in 6-12 months, if thecolposcopists do not see a clear indication of a lesion that should bebiopsied. This would decrease the number of blind biopsies that arecurrently being performed, decreasing screening costs and increasinghealth care quality.

The inventors have developed a method that can be used to triage womenwith HPV+ abnormal Pap smears who have been referred to colposcopy.Participants were women living in Chile and Puerto Rico with no cervicalintraepithelial lesions or malignancy and women with abnormal cervicalbiopsies-CIN2+. The inventors used methylation arrays during theDiscovery phase, followed by bisulfite sequencing and quantitativeMethylation Specific PCR (qMSP) during the Validation phase. Usingcustom sequence capture pools of baits, the inventors pulled downgenomic and bisulfite converted high-risk HPV DNA before library prepfor Next Generation Sequencing (NGS) in 454 and MiSeq instruments,respectively. Using the methylation arrays results, they optimized aSyber Greeen qPCR assay to detect high risk HPV DNA and a qMSPprimer-probe set to quantify promoter methylation of ZNF516, FKBP6, andINTS1 in the host and the L1 gene in the HPV genome, in liquid prep andcirculating cell free DNA (ccfDNA) in plasma and urine.

Additionally, we have quantified the circulating HPV methylome in urine,using a custom sequence capture approach, which allows for multiplexedmassively parallel sequence of clinical sample, followed by qMSPverification. Our results also show that qMSP quantification of HPV16-L1methylation is a surrogate of genome-wide HPV16 methylation, which couldlead to high-throughput testing of HPV16-L1 methylation by qMSP, digitalPCR or multiplexed massively parallel sequencing. Furthermore, theHPV16-L1 methylation assay discriminates ccfDNA in urine from women withcervical dysplasia when compared to women that do not have cervicaldysplasia.

Cervical cancer affects more than 1,000,000 women worldwide. Around470,000 new cases of cervical cancer are detected annually, mostly indeveloping nations, among which approximately half will die (42).Persistent mucosal infection with an oncogenic (high risk) HPV genotypeis the most significant cause of cervical dysplasia and carcinoma (43).Only 14 of the genotypes are considered pathogenic or high-risk (44).Multiple studies have linked genotypes 16, 18, 31, 33, 35, 39, 45, 51,52, 56, 58, 59, 66, and 68 to disease progression (45). Patients with apersistent infection with one of these types have an increased risk fordeveloping severe dysplasia or cervical carcinoma (46).

Co-testing with cytology and HPV at 5-year intervals is now thepreferred or acceptable strategy for cervical cancer screening for womenaged 30-64 years in the US. Clinical management forHPV-positive/Pap-negative women, however, is not firmly established. Inaddition, there is increased resistance from the medical community toaccept HPV-PAP co-testing for cervical cancer, due to the complex riskpatterns associated to positive, negative, and undetermined cytologywith positive and negative HPV results.

Clinical detection of HPV is typically performed by in-vitro diagnosticassays that detect viral genomic DNA, specifically the L1 gene, onmucosa samples collected by cervical scraping. However, because HPVinfections are very common and because most women will clear HPVinfections within 6 to 12 months, the presence of HPV DNA does not meanthat cervical dysplasia or cervical cancer is present or that theinfection will persist and the patient will progress to cervical cancer(13). Furthermore, cervical cancer screening programs currently in useare inefficient at identifying individuals at risk for disease,requiring multiple visits over a women's lifetime, which is costly andcumbersome (47). New methods for cervical cancer screening are needed toprovide accurate, efficient and cost-effective ways of identifying womenat risk for cervical cancer.

Methods

Patient Samples

Cervical brush, liquid-based cytology, serum/plasma and urine sampleswere obtained from collaborators Chile and Puerto Rico, under the JohnsHopkins University School of Medicine Institutional Review Board (IRB)approved protocol #NA 00020633. The IRB of the Doctor Hernán HenríquezAravena (HHA) tertiary care regional hospital, in Temuco, Chile and IRBof the University of Puerto Rico School of Medicine also approved thisprotocol.

Sample Collection and Processing

Retrospective Cohort

DNA was isolated from the cervical epithelium and biopsy samples,genotyped for HPV with the Reverse Line Blot assay in Chile and sent toJohns Hopkins School of Medicine for epigenome-wide studies, aspreviously described (Epigenetics, 9, 308 (Feb. 1, 2014)).

Prospective Cohort

Samples were collected, flash frozen and sent to Johns Hopkins School ofMedicine for DNA extraction with phenol/chloroform method, bisulfiteconversion NGS, qPCR and qMSP analyses as described below.

Nucleic Acid Extraction

Cervical cytobrush, liquid-based cytology, and plasma/serum samples(n=40) were centrifuged and their pellets were digested with 1% SDS and20 μg/mL proteinase K (Sigma) at 48° C. for 48 h, followed byphenol/chloroform extraction and ethanol precipitation.

Identification of Methylation Biomarkers of CIN2+ in Liquid-BasedCytology

We used quantitative Methylation Specific PCR (qMSP) to examine theassociation between CIN2+ biopsies and methylation of three genes(ZNF516, FKBP6, and INTS1) in cervical brush epithelium from aretrospective cohort, in which these genes are associated with cervicalcancer and abnormal cytology (18). We verified the association betweenCIN2+ biopsies and methylation of ZNF516, FKBP6, INTS1 and HPV16-L1 inliquid-based cytology samples obtained from an independent prospectivecohort.

Quantitative Methylation-Specific PCR (qMSP)

The methylation status of ZNF516, FKBP6, INTS1 and HPV16-L1 in bisulfitemodified DNA from liquid-based cytology samples, plasma/serum and urine,was quantified with fluorescence-based quantitative methylation-specificPCR (qMSP) as described previously (Oncology Reports 32, 505 (August,2014), Epigenetics, 9, (May 1, 2014)). Briefly, bisulfite converted DNAwas used as template for fluorescence-based real-time PCR. Bisulfitesequencing (BS) was performed to determine the methylation status of thenormal and tumor tissues prior to MSP. Bisulfite-treated DNA wasamplified using BS primer sets for a 5′ region within 800 bp of the TSSthat included at least part of a CpG Island. The primer sequences didnot contain CpG dinucleotides in order to obtain unbiased sequencing PCRproducts. Each amplified DNA sample was sequenced using forward orreverse primers. After verifying with bisulfite sequencing that we hadlocated a suitable area in the promoter region for qMSP validation, MSPprimers and qMSP probes were designed to specifically amplify thisregion in the candidate gene promoters. Primers and probes were testedon positive (in vitro methylated bisulfite converted DNA) and negativecontrols (genomic unmethylated bisulfite converted DNA) to ensureamplification of the desired product and non-amplification ofunmethylated DNA, respectively. Primer and probe sequences are providedbelow.

Fluorogenic PCR reactions were performed in duplicates in a reactionvolume of 20 μL that contained 3 μL of bisulfite-modified DNA; 600 nMconcentrations of forward and reverse primers; 200 nM probe; 0.6 U ofplatinum Taq polymerase (Invitrogen, Frederick, Md.); 200 μMconcentrations each of dATP, dCTP, dGTP and dTTP; and 6.7 mM MgCl2.Amplifications were performed using the reaction profile: 95° C. for 3min, followed by 50 cycles at 95° C. for 15 s and 60° C. for 1 min in a7900HT sequence detector (Applied Biosystems) and were analyzed by asequence detector system software (SDS 2.4; Applied Biosystems).

Primers and Probes

Primers and probes sequences for the HPV16-L1 gene region and for theccfDNA amplicons isolated from ccfDNA are shown below.

HPV16-L1 F: (SEQ ID NO: 1) TATAGCGGTTGGTTTGGGTTTGTG R: (SEQ ID NO: 2)ACATTCTCTATTATCCACACCTACA Probe: (SEQ ID NO: 3)/56-FAM/AGGTGTTGAGGTAGGTCGTGG-TAMRA-SpccfDNA Primers and Probes(Probes are: 5′/56-FAM/−/ZEN/−/3IABkFQ/3′)

FKBP6 F: (SEQ ID NO: 4) ATATTTCGTATTTTATCGCG R: (SEQ ID NO: 5)ATCGTTTCGTTCCAACCG Probe: (SEQ ID NO: 6) CGACCCTAACCCTCGCGAACTCTA ZNF516F: (SEQ ID NO: 7) ACGGTGAGGTATGTATACG R: (SEQ ID NO: 8)ACTCGAAACCCTCAAAACG Probe: (SEQ ID NO: 9) AACGCCAAACCTCACCGTCGTACG INTS1F: (SEQ ID NO: 10) CGTTTTTCGTCGTCGTTTTA R: (SEQ ID NO: 11)AAACAAAAAAAATAACCGACGAT Probe: (SEQ ID NO: 12) TATAACCTCCGCCCTCCCTCCCTAUrine ccfDNA Extraction and Assessment

We adsorbed cell-free nucleic acids, on a Q-Sepharose anion-exchangeresin followed by a silica-based elution with LiCl, from urine samples(10 mL) provided by 31 women with normal cervical cytology and 65 womenwith abnormal cervical cytology and biopsy: CIN1 (n=43) and CIN2-3(n=22). ccfDNA samples were quantitated using the Quant-iT PicoGreendsDNA reagent kit (Invitrogen, Life Technologies) and QuantiFluor STspectrofluorometer (Promega). Concentrations were calculated based onreadings obtained from Lambda DNA standards. Quality (fragment size)assessment of the ccfDNA samples was performed by High Sensitivity DNALab Chip analysis on a BioAnalyzer 2100 (Agilent). The urine ccfDNAsamples selected for further processing showed size profiles in the50-300 bp range, and exhibited low molecular weight DNA content.

ccfDNA hrHPV SYBR® Green Real-Time PCR

Validation of successful capture was assessed by the ccfDNA hrHPV qPCRassay on an Applied Biosystems Systems Prism 7500 Sequence DetectionSystem. We designed SYBR® green quantitative PCR amplification assays ofBeta-actin and the HPV E1 region that was used for the ccfDNA hrHPVcapillary electrophoresis test (J. Clin. Microbiol. 52, 187 (January,2014), J. Clin. Virol. (May 2, 2014)). This HPV E1 region is common to13 high-risk HPV types (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58,59 and 68). The resulting amplicon is 93 bp. Beta-actin (β-actin), ahousekeeping gene, was used as an indicator for successful extraction ofan equivalent length of viral DNA targets. Serial dilutions of DNAisolated from cervical carcinoma cell line CaSki (ATCC® CRL-1550™, 600integrated HPV16 copies) were run in parallel as positive controls.Primers were obtained from Invitrogen (Carlsbad, Calif.).

Custom Dual Sequence Capture Assay

HPV-specific, biotinylated, long oligonucleotide probes were designed,synthesized, and pooled for target selection and enrichment utilizing adual capture approach (Roche/NimbleGen SeqCap EZ Choice Library). Probeswere designed to capture the complete HPV genome and well-characterizedvariants representing 12 clinically relevant high-risk HPV types thathave been associated with cervical cancer (HPV types 16, 18, 31, 33, 35,39, 45, 52, 56, 58, 59 and 68b). The dual capture approach features twosequential captures of HPV target regions, with the output of the firstcapture amplified and used as targets for a second capture. The goal ofthis dual capture approach was a boost in both enrichment andspecificity of HPV targets for deep sequencing.

Library preparation was performed using reagents in the GS FLX TitaniumRapid Library Preparation kit (Roche). To benchmark the assay we firstworked with HPV positive cell lines: HeLa (HPV 18) and CSCC7 (HPV16).For these cell line samples, 250 ng genomic DNA was fragmented bynebulization, prior to ligation of double stranded, Rapid LibraryMultiplex Identifier (RL MID) adaptors, which added unique 10 base pairsequence tags to each library enabling multiplexing for sequencing,according to standard Roche protocol.

Methods in the Roche Rapid Library preparation manual were adapted towork with the fragmented ccfDNA obtained from urine and optimized toprepare libraries with as little as 30 nanograms of DNA (range=30-100nanograms). Briefly, End-Polishing and A-tailing of fragments wasperformed, followed by ligation of the RL MID adaptors. Subsequently,two rounds of purification to remove un-ligated adaptors and adaptordimers were performed on the fragments using AmPure XP beads accordingto standard Agencourt protocol. Quantitation of the library versus aFAM-labeled standard was performed using a QuantiFluor STspectrofluorometer (Promega). Quality assessment of the Library wasperformed by High Sensitivity DNA Lab Chip analysis on a BioAnalyzer2100 (Agilent). A pre-capture amplification of the library was performedwith ligation-mediated PCR (LM-PCR) for 12 cycles using primerscomplementary to the adaptors, followed by two rounds of hybridizationto the HPV-specific SeqCap EZ Choice Library. Each hybridizationincluded enhancer oligonucleotides as well as Cot-1 Blocking DNA. Aftereach hybridization step, a Streptavidin-coated, magnetic bead-basedcleanup was performed, and the captured DNA was re-amplified by LM-PCR(5 cycles Post-Cap1, 15 cycles Post-Cap2). Subsequent to both Post-CapLM-PCRs, purification was performed using the QiaQuick PCR purificationkit (Qiagen). After Post-Cap2 LM-PCR, HPV-enriched ccfDNAs werequantitated by PicoGreen fluorescent assay and quality was assessed byDNA High Sensitivity Lab Chip analysis on the BioAnalyzer 2100.

A calculation of fold-enrichment was based on Ct values of captured vs.non-captured LM-PCR products in comparison with positive and negativecontrols. The amplified, HPV-enriched cell line DNAs and ccfDNAs werethen diluted to a normalized concentration of 1×E08 molecules permicroliter, pooled and processed for multiplexed sequencing on the GSJunior system (Roche).

Validation of successful capture was assessed by a XEN-HPV qPCR Sybrgreen assay on an Applied Biosystems Systems 7500 Sequence DetectionSystem. A calculation of fold-enrichment was based on Ct values ofcaptured vs. non-captured LM-PCR products in comparison with positiveand negative controls. The amplified, HPV-enriched cell line DNAs andccfDNAs were then diluted to a normalized concentration of 1×E08molecules per microliter, pooled and processed for multiplexedsequencing on the GS Junior system (Roche).

RT-PCR Assay

A Perkin-Elmer/ABI 7900 thermocycler was used to run SYBR greenquantitative PCR amplification assays for β-actin and the HPV E1 regionthat was used for the ccfDNA HPV capillary electrophoresis test. ThisccfDNA HPV qPCR assay amplifies an HPV E1 region common to 13 high-riskHPV types (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68).The resulting amplicon is 93 bp. Primers were obtained from Invitrogen(Carlsbad, Calif.).

Sequencing on the GS Junior System

Processing for sequencing was according to Lib-L emPCR AmplificationMethod and Sequencing Method manuals, GS Titanium Series (Roche). Amodification of reduced amplification primer (1:10 dilution) in theemPCR step was performed due to the small fragment size range. The DNAcopy (fragment) to bead ratio for emPCR was 0.5. Sequence runs wereperformed on the GS Junior System.

Following completion of the sequencing runs, signal processing wasperformed, followed by QC analyses. Roche GS RunBrowser softwaredeconvoluted the data, assigned each read to the appropriate library,and was used to assess QC metrics (% Keypass wells, % pass filtering,Average Read length, and Total bases). GS Reference Mapper, from theRoche GS Analysis Software Suite, was used for initial alignment to theHPV 16 and HPV 18 reference genomes.

Detailed analysis was performed as described below.

Bioinformatics Analyses for GS Junior Output Reads.

To determine the HPV genotype composition of the patient samples, wesearched the GS Junior 454 reads from each ccfDNA sample against thenon-redundant database of HPV reference types compiled from the twelvehigh-risk and nine low-risk HPV reference genomes and the largercollection of PapillomaVirus Episteme (PAVE) genome database (48).Sequences were aligned to the reference genome database with thesoftware sim4db (49), retaining only the best alignment covering 50% ormore of the query sequence. To resolve the remaining reads, we firstsearched the human genome with the program bowtie (39), allowing forpartial matches (option ‘--local’), and then the database of NCBIbacterial reference genomes, using the tool blast (40). Following thistiered mapping approach, only a small number of reads were stillunclassified, of which a small number (<100) were linker contaminants,whereas the others could potentially represent novel HPV genotypes andother viruses or bacteria.

Custom Methylated HPV Sequence Capture and Sequencing

To quantify the HPV methylome by HPV type in ccfDNA we developed amethylated HPV sequence method (HPV ccfDNA Meth-seq) using customdesigned baits (Table 5). The Sure Select Methyl-Seq Target Enrichment(Agilent) workflow, developed to capture the Human Methylome with astarting DNA amount of 3 μg, was optimized to capture the HPV methylomeof 12 high-risk HPV types that have been associated with cervical cancer(HPV types 16, 18, 31, 33, 35, 39, 45, 52, 56, 58, 59 and 68b). Briefly,the Sure Select Methyl-Seq Library Prep Kit was used for End-Polishingand A-tailing of sheared DNA (150-200 bp) fragments, and subsequentligation of methylated adaptors. These libraries were hybridized totarget enrichment RNA baits in the custom SureSelect HPV Methyl-Seq baitlibrary and hybrids were bound to streptavidin beads for enrichment. Thetarget enriched gDNA library was then bisulfate treated and subsequentlyamplified by PCR and index tagged by a second PCR. The indexed,bisulfate-treated and enriched library was pooled, diluted to 2 nM, andsubmitted to the JHMI Synthesis and Sequencing Facility for sequencingon the Ilumina MiSeq system. The library was denatured using 0.2 N NaOHand diluted to a loading concentration of 15 pM. Sequencing wasperformed using a 2×150 MiSeq Reagent Kit v2 (300 cycle) with the runperformed as a 2×100 paired-end run. The sample was loaded with a 5%PhiX control spike-in to account for the low diversity of the samplelibrary. FASTQ files were generated and analyzed as follows.

Analysis of Sequencing Data

FASTQC version 0.11.3 was used for quality control of all the paired-endreads to assess per sequence base quality, per tile sequence quality,per sequence quality scores, per base sequence content, per sequence GCcontent, per base N content, sequence length distribution, sequenceduplication levels, overrepresented sequences, adapter and kmer content.Reads were trimmed using Trim Galore v0.3.7. Default parameters wereused, and one base pair was trimmed off at the end of all paired-endreads to improve paired-end mapping. If adapter contamination wasobserved, the standard Illumina adapters were trimmed off at the end ofall paired-end reads. FASTQC post-trimming was rerun to perform qualitycontrol to ensure sure that the trimming step did not produce anyadverse side effects.

Bioinformatics Analyses of Bisulfite-Converted Reads

Briefly, for alignment purposes Bismark converts all C's to T's (inforward reads) and all G's to A's (in reverse reads) prior to mappingand maps these in silico converted reads, to both a C-to-T and G-to-A insilico-converted genome. After successful alignment it replaces the T'sand A's back to their original bases in all converted reads and comparesit to the original reference genome to deduce methylated cytosines.Default parameters were used with the exception that “bowtie2 and 1mismatch” was allowed during the alignment, retaining only the uniquematches (default) and a seed length of 32. PCR duplicates were removedfrom the mapped reads using the “de-duplicate Bismark” routine. Afterrunning Bismark, post-alignment quality control was performed usingSamtools version 0.1.19 and BamUtil version 1.0.12. Bismark divides allcytosines into four categories: cytosines followed by guanines (CpGs),cytosines followed by non-guanines followed by guanines (CHGs),cytosines followed by at least two non-guanines (CHHs), and cytosinesfollowed by N's (CNs). Analysis for the current study focused on CpGs.The Default Bismark methylation extractor routine was used with theexception of --paired-end, --no-overlap, and minimum coverage of atleast 1 read to extract all CpGs in individual samples.

Sequence similarity among HPV reference genomes could potentially impactthe analysis, by disqualifying reads that map to several genomes. Toeliminate or reduce these effects, we employed a two stage mappingprocedure: i) we first aligned the reads against the entire genomedatabase, using the unique matches to select those genotypes representedin the samples, and then ii) re-aligned the reads against the subset ofgenomes that were detected in that sample. Methylation maps for theHPV16 genome and restricted to the L1 region were produced with dataproduced by the Methylation Extractor from the Bismark suite.

REFERENCES

All publications, patent applications, patents, and other referencesmentioned in the specification are indicative of the level of thoseskilled in the art to which the presently disclosed subject matterpertains. All publications, patent applications, patents, and otherreferences are herein incorporated by reference to the same extent as ifeach individual publication, patent application, patent, and otherreference was specifically and individually indicated to be incorporatedby reference. It will be understood that, although a number of patentapplications, patents, and other references are referred to herein, suchreference does not constitute an admission that any of these documentsforms part of the common general knowledge in the art. In case of aconflict between the specification and any of the incorporatedreferences, the specification (including any amendments thereof, whichmay be based on an incorporated reference), shall control. Standardart-accepted meanings of terms are used herein unless indicatedotherwise. Standard abbreviations for various terms are used herein.

In case of a conflict between the specification and any of theincorporated references, the specification (including any amendmentsthereof, which may be based on an incorporated reference), shallcontrol. Standard art-accepted meanings of terms are used herein unlessindicated otherwise. Standard abbreviations for various terms are usedherein.

-   1. J. C. Gage et al., The low risk of precancer after a screening    result of human papillomavirus-negative/atypical squamous cells of    undetermined significance papanicolaou and implications for clinical    management. Cancer cytopathology, (Jul. 9, 2014).-   2. M. Schiffman et al., Human papillomavirus testing in the    prevention of cervical cancer. Journal of the National Cancer    Institute 103, 368 (Mar. 2, 2011).-   3. G. Y. Ho, R. Bierman, L. Beardsley, C. J. Chang, R. D. Burk,    Natural history of cervicovaginal papillomavirus infection in young    women. The New England journal of medicine 338, 423 (Feb. 12, 1998).-   4. M. R. McCredie et al., Natural history of cervical neoplasia and    risk of invasive cancer in women with cervical intraepithelial    neoplasia 3: a retrospective cohort study. The lancet oncology 9,    425 (May, 2008).-   5. V. Cogliano et al., Carcinogenicity of human papillomaviruses.    The lancet oncology 6, 204 (April, 2005).-   6. P. E. Castle, J. C. Gage, C. M. Wheeler, M. Schiffman, The    clinical meaning of a cervical intraepithelial neoplasia grade 1    biopsy. Obstetrics and gynecology 118, 1222 (December, 2011).-   7. J. C. Gage et al., Comparison of the cobas Human Papillomavirus    (HPV) test with the hybrid capture 2 and linear array HPV DNA tests.    Journal of clinical microbiology 50, 61 (January, 2012).-   8. D. Saslow et al., American Cancer Society, American Society for    Colposcopy and Cervical Pathology, and American Society for Clinical    Pathology screening guidelines for the prevention and early    detection of cervical cancer. American journal of clinical pathology    137, 516 (April, 2012).-   9. H. A. Katki et al., Five-year risks of CIN 3+ and cervical cancer    among women who test Pap-negative but are HPV-positive. Journal of    lower genital tract disease 17, S56 (April, 2013).-   10. L. S. Massad et al., 2012 updated consensus guidelines for the    management of abnormal cervical cancer screening tests and cancer    precursors. Obstetrics and gynecology 121, 829 (April, 2013).-   11. J. C. Gage et al., Reassurance against future risk of precancer    and cancer conferred by a negative human papillomavirus test.    Journal of the National Cancer Institute 106, (August, 2014).-   12. H. A. Katki et al., Five-year risk of recurrence after treatment    of CIN 2, CIN 3, or AIS: performance of HPV and Pap cotesting in    posttreatment management. Journal of lower genital tract disease 17,    S78 (April, 2013).-   13. M. Schiffman, N. Wentzensen, Human papillomavirus infection and    the multistage carcinogenesis of cervical cancer. Cancer    epidemiology, biomarkers & prevention: a publication of the American    Association for Cancer Research, cosponsored by the American Society    of Preventive Oncology 22, 553 (April, 2013).-   14. A. F. Fernandez et al., The dynamic DNA methylomes of    double-stranded DNA viruses associated with human cancer. Genome    research 19, 438 (March, 2009).-   15. L. Mirabello et al., Elevated methylation of HPV16 DNA is    associated with the development of high grade cervical    intraepithelial neoplasia. International journal of cancer. Journal    international du cancer 132, 1412 (Mar. 15, 2013).-   16. N. Wentzensen et al., Methylation of HPV18, HPV31, and HPV45    genomes and cervical intraepithelial neoplasia grade 3. Journal of    the National Cancer Institute 104, 1738 (Nov. 21, 2012).-   17. L. Mirabello et al., Methylation of human papillomavirus type 16    genome and risk of cervical precancer in a Costa Rican population.    Journal of the National Cancer Institute 104, 556 (Apr. 4, 2012).-   18. C. Sun, L. L. Reimers, R. D. Burk, Methylation of HPV16 genome    CpG sites is associated with cervix precancer and cancer.    Gynecologic oncology 121, 59 (April, 2011).-   19. N. Vasiljevic, D. Scibior-Bentkowska, A. Brentnall, J.    Cuzick, A. Lorincz, A comparison of methylation levels in HPV18,    HPV31 and HPV33 genomes reveals similar associations with cervical    precancers. Journal of clinical virology: the official publication    of the Pan American Society for Clinical Virology 59, 161 (March,    2014).-   20. N. Vasiljevic, D. Scibior-Bentkowska, A. R. Brentnall, J.    Cuzick, A. T. Lorincz, Credentialing of DNA methylation assays for    human genes as diagnostic biomarkers of cervical intraepithelial    neoplasia in high-risk HPV positive women. Gynecologic oncology 132,    709 (March, 2014).-   21. A. Lendvai et al., Genome-wide methylation profiling identifies    hypermethylated biomarkers in high-grade cervical intraepithelial    neoplasia. Epigenetics: official journal of the DNA Methylation    Society 7, 1268 (November, 2012).-   22. J. J. Eijsink et al., A four-gene methylation marker panel as    triage test in high-risk human papillomavirus positive patients.    International journal of cancer. Journal international du cancer    130, 1861 (Apr. 15, 2012).-   23. P. Brebi et al., Genome-wide methylation profiling reveals Zinc    finger protein 516 (ZNF516) and FK-506-binding protein 6 (FKBP6)    promoters frequently methylated in cervical neoplasia, associated    with HPV status and ethnicity in a Chilean population. Epigenetics:    official journal of the DNA Methylation Society 9, 308 (Feb. 1,    2014).-   24. A. R. Brentnall et al., A DNA methylation classifier of cervical    precancer based on human papillomavirus and human genes.    International journal of cancer. Journal international du cancer    135, 1425 (Sep. 15, 2014).-   25. K. Mendez et al., Urine-based human papillomavirus DNA testing    as a screening tool for cervical cancer in high-risk women.    International journal of gynaecology and obstetrics: the official    organ of the International Federation of Gynaecology and Obstetrics    124, 151 (February, 2014).-   26. V. V. Sahasrabuddhe et al., Comparison of human papillomavirus    detections in urine, vulvar, and cervical samples from women    attending a colposcopy clinic. Journal of clinical microbiology 52,    187 (January, 2014).-   27. C. Payan et al., Human papillomavirus quantification in urine    and cervical samples by using the Mx4000 and LightCycler general    real-time PCR systems. Journal of clinical microbiology 45, 897    (March, 2007).-   28. A. Vorsters et al., Optimization of HPV DNA detection in urine    by improving collection, storage, and extraction. European journal    of clinical microbiology & infectious diseases: official publication    of the European Society of Clinical Microbiology, (Jun. 12, 2014).-   29. A. Vorsters et al., Detection of human papillomavirus DNA in    urine. A review of the literature. European journal of clinical    microbiology & infectious diseases: official publication of the    European Society of Clinical Microbiology 31, 627 (May, 2012).-   30. A. Ducancelle et al., Interest of Human Papillomavirus DNA    quantification and genotyping in paired cervical and urine samples    to detect cervical lesions. Archives of gynecology and obstetrics,    (Mar. 13, 2014).-   31. A. V. Lichtenstein, H. S. Melkonyan, L. D. Tomei, S. R. Umansky,    Circulating nucleic acids and apoptosis. Annals of the New York    Academy of Sciences 945, 239 (September, 2001).-   32. Y. H. Su et al., Transrenal DNA as a diagnostic tool: important    technical notes. Annals of the New York Academy of Sciences 1022, 81    (June, 2004).-   33. E. M. Shekhtman et al., Optimization of transrenal DNA analysis:    detection of fetal DNA in maternal urine. Clinical chemistry 55, 723    (April, 2009).-   34. H. S. Melkonyan et al., Transrenal nucleic acids: from proof of    principle to clinical tests. Annals of the New York Academy of    Sciences 1137, 73 (August, 2008).-   35. A. Cannas et al., Mycobacterium tuberculosis DNA detection in    soluble fraction of urine from pulmonary tuberculosis patients. The    international journal of tuberculosis and lung disease: the official    journal of the International Union against Tuberculosis and Lung    Disease 12, 146 (February, 2008).-   36. V. V. Sahasrabuddhe et al., Evaluation of clinical performance    of a novel urine-based HPV detection assay among women attending a    colposcopy clinic. Journal of clinical virology: the official    publication of the Pan American Society for Clinical Virology, (May    2, 2014).-   37. M. Steinau et al., Performance of commercial reverse line blot    assays for human papillomavirus genotyping. Journal of clinical    microbiology 50, 1539 (May, 2012).-   38. I. S. Park et al., Characterization of the methylation patterns    in human papillomavirus type 16 viral DNA in head and neck cancers.    Cancer prevention research 4, 207 (February, 2011).-   39. B. Langmead, S. L. Salzberg, Fast gapped-read alignment with    Bowtie 2. Nature methods 9, 357 (April, 2012).-   40. S. F. Altschul et al., Gapped BLAST and PSI-BLAST: a new    generation of protein database search programs. Nucleic acids    research 25, 3389 (Sep. 1, 1997).-   41. P. M. Thompson et al., The ENIGMA Consortium: large-scale    collaborative analyses of neuroimaging and genetic data. Brain    imaging and behavior 8, 153 (June, 2014).-   42. S. Beaudenon, J. M. Huibregtse, HPV E6, E6AP and cervical    cancer. BMC Biochem 9 Suppl 1, S4 (2008).-   43. D. Dehn, K. C. Torkko, K. R. Shroyer, Human papillomavirus    testing and molecular markers of cervical dysplasia and carcinoma.    Cancer 111, 1 (Feb. 25, 2007).-   44. S. K. Kjaer et al., Type specific persistence of high risk human    papillomavirus (HPV) as indicator of high grade cervical squamous    intraepithelial lesions in young women: population based prospective    follow up study. BMJ 325, 572 (Sep. 14, 2002).-   45. J. Monsonego et al., Cervical cancer control, priorities and new    directions. Int J Cancer 108, 329 (Jan. 20, 2004).-   46. K. S. Cuschieri, M. J. Whitley, H. A. Cubie, Human    papillomavirus type specific DNA and RNA persistence—implications    for cervical disease progression and monitoring. J Med Virol 73, 65    (May, 2004).-   47. A. J. Brown, C. L. Trimble, New technologies for cervical cancer    screening. Best practice & research. Clinical obstetrics &    gynaecology 26, 233 (April, 2012).-   48. K. Van Doorslaer et al., The Papillomavirus Episteme: a central    resource for papillomavirus sequence data and analysis. Nucleic    acids research 41, D571 (January, 2013).-   49. B. Walenz, L. Florea, Sim4db and Leaff: utilities for fast batch    spliced alignment and sequence indexing. Bioinformatics 27, 1869    (Jul. 1, 2011).-   50. F. Krueger, S. R. Andrews, Bismark: a flexible aligner and    methylation caller for Bisulfite-Seq applications. Bioinformatics    27, 1571 (Jun. 1, 2011).

That which is claimed:
 1. A method for triaging a human papillomavirus(HPV)-positive woman into colposcopy, the method comprising: (a)selecting a HPV-positive woman testing positive for one or more highrisk types of HPV; (b) obtaining nucleic acid from a test sample from aliquid prep, plasma, serum or urine of the selected HPV-positive woman;(c) performing bisulfite modification to the nucleic acid from the testsample to produce a bisulfite modified nucleic acid; (d) determining inthe bisulfite modified nucleic acid from (c) a promoter methylationlevel of the promoter regions of the group of genes comprising ZNF516,FKBP6, and INTS1 and HPV gene HPV16-L1 using quantitative real-timemethylation specific PCR (QMSP) and primers and/or probes are selectedfrom the group consisting of SEQ ID NOS: 1-12 that exhibit increasedpromoter methylation in women having CIN12+ lesions as compared to womenhaving no intraepithelial lesions or malignancy (NILM); (e) triaging theHPV-positive woman into colposcopy when the level of promotermethylation of the group of genes is increased relative to the level ofpromoter methylation of the group of genes in a reference sampleobtained from women having NILM; and (f) performing a colposcopy.
 2. Themethod of claim 1, wherein selecting the HPV-positive woman testingpositive for one or more high risk types of HPV comprises determiningwhether the nucleic acid is homologous to one or more high risk types ofHPV.
 3. The method of claim 2, wherein determining whether the nucleicacid is homologous to one or more high risk types of HPV comprisesperforming at least one HPV detection assay selected from the groupconsisting of nucleic acid sequencing, PCR, a HPV genotyping assay, amicroarray assay, and a mRNA based assay.
 4. The method of claim 3,wherein determining whether the nucleic acid is homologous to one ormore high risk types of HPV comprises: (a) sequencing the nucleic acidto produce a nucleotide sequence; (b) performing a sequence alignmentbetween the nucleotide sequence and the nucleotide sequence of the oneor more high risk types of HPV; and (c) determining the percentagesequence identity between the nucleotide sequence and the nucleotidesequence of the one or more high risk types of HPV.
 5. The method ofclaim 4, wherein the one or more high risk types of HPV are selectedfrom the group consisting of HPV16, HPV18, HPV31, HPV33, HPV35, HPV39,HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, and HPV68.
 6. The method ofclaim 1, wherein the nucleic acid comprises DNA.
 7. The method of claim1, wherein the nucleic acid comprises TrDNA.
 8. The method of claim 1,wherein the nucleic acid is from about 150 to about 250 base pairs. 9.The method of claim 8, wherein the HPV-positive woman is furtherselected on the basis of abnormal cytology.
 10. The method of claim 9,wherein the HPV-positive woman has had a negative, positive, orinconclusive Pap smear.
 11. The method of claim 1, wherein the methodhas a specificity of at least 60%.
 12. The method of claim 1, whereinthe method has a sensitivity of at least 90%.
 13. The method of claim 1,wherein the method has a positive predictive value (PPV) of at least52%.
 14. The method of claim 1, wherein the method has a negativepredictive value (NPV) of at least 90%.
 15. The method of claim 1,further comprising enriching the nucleic acid from the test samplebefore determining the promoter methylation level; wherein enriching thenucleic acid from the test sample comprises: (1) preparing a library ofthe nucleic acid from the test sample; (2) amplifying the library usingPCR to form a pre-capture PCR library; (3) hybridizing the pre-capturePCR library to a custom-designed pool of HPV-specific and human-specificcapture probes to form a post-capture PCR library; (4) amplifying thepost-capture PCR library to produce enriched nucleic acid; and (5)optionally repeating steps (3) and (4).
 16. The method of claim 1,further comprising recommending treatment and/or treating the woman,wherein the treatment is selected from the group consisting of removalof precancerous lesions, radiation treatment, surgery, and chemotherapy.